In order to understand the spatial-temporal distribution and ecological risk of antibiotics in the soil of an agricultural watershed in the Three Gorges Reservoir area, the topsoil samples were collected at 26 sites in the Wangjiagou small watershed, Fuling District, Chongqing in spring, summer, autumn, and winter of 2022, and 21 antibiotics with five classes were determined using solid phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry. The content levels and spatial-temporal distribution of antibiotics were analyzed, the correlations between antibiotic contents and soil physicochemical factors were discussed, and the potential ecological risk of antibiotics in the soil was evaluated using the risk quotient method. The results showed that the detection rates of 21 antibiotics were 0-100% with the range of ND-219.5 μg·kg -1 , and those of tetracyclines (TCs), quinolones (FQs), and chloramphenicols (CAPs) reached 100% in all four seasons. The total antibiotic content ranged from 14.35 to 504.1 μg·kg -1 with the average value of 149.7 μg·kg -1 , and the average contents of the five classes of antibiotics showed a decreasing trend of TCs (77.95 μg·kg -1 ), FQs (34.96 μg·kg -1 ), CAPs (28.14 μg·kg -1 ), sulfonamides (SAs, 7.15 μg·kg -1 ), and macrolides (MLs, 1.48 μg·kg -1 ). The antibiotic contents of soils during autumn and winter were significantly higher than those in summer ( P < 0.05) and showed an overall variation trend of first decreasing and then increasing with the season and a distribution situation of "low in the west and high in the east." The sites with high antibiotic contents were mainly concentrated in vegetable fields, residential areas, and dry lands. In spring soils, the contents of tylosin (TYL) ( P < 0.01), tetracycline (TC) ( P < 0.01), doxycycline (DXC), and sulfamethodiazine (SMR) ( P < 0.05) were significantly negatively correlated with soil pH. In summer soils, total phosphorus (TP) had significantly positive effects on the contents of sulfamethoxazole (SFZ) and TC ( P < 0.05) and had extremely significant positive effects on sulfamethoxazole (SMX), SMR, and ofloxacin (OFL) ( P < 0.01). Total nitrogen (TN) had significantly positive effects on the contents of SMX ( P < 0.01) and SMR ( P < 0.05), and soil organic matter (SOM) had significantly positive effects on the contents of TC and enrofloxacin (ENR) ( P < 0.05). However, ciprofloxacin (CIP) ( P < 0.001), sulfampyridine (SPD), norfloxacin (NOR), and TYL contents ( P < 0.05) were significantly negatively correlated with soil pH. In autumn soils, TN significantly negatively affected CIP content ( P < 0.05); the contents of sulfadiazine (SDZ), TC, and DXC were negatively affected by soil pH ( P < 0.05); and cation exchange capacity (CEC) significantly positively affected the contents of TC and SFZ ( P < 0.05). In winter soils, TP had a significantly positive effect on TYL content, and TN had a significantly positive effect on SFZ content ( P < 0.05) and an extremely positive effect on sulfamethoxine (SMM) ( P < 0.001). Chlorotetracycline (CTC) ( P < 0.01), CIP ( P < 0.01), thiamphenicol (TAP) ( P < 0.001), chloramphenicol (CAP) ( P < 0.001), oxytetracycline (OTC) ( P < 0.001), and sulfadimidine (SM2) ( P < 0.05) were significantly negatively correlated with soil pH, and CEC had a significantly negative effect on SDZ content ( P < 0.05). The soils in the Wangjiagou agricultural small watershed were mainly threatened by SMX, SDZ, TC, DXC, erythromycin, NOR, OFL, CIP, and ENR, and their ecological risks should not be ignored. more...