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Kaili Red Sour Soup (RSS) is a traditional fermented vegetable sauce made from red chili pepper and tomato, through special two-round anaerobic fermentations in southwest China, however, its physicochemical, microbial and flavor characteristics remain unclear. The pH value, total titratable acidity, amino acid nitrogen and nitrite of repined Kaili RSS were determined to be 3.26, 27.34 g/kg, 3.04 mg/kg and3.04 mg/kg, respectively. HPLC spectra showed 7 organic acids in RSS, and lactic acid (24.71 g/kg) and acetic acid (6.41 g/kg) mainly contribute to sour tastes. 15 Amino acids were found in RSS, and alanine (73.19 mg/100g) directly contributed to RSS's umami. The results of GC-MS illustrated totally 76 volatile organic compounds including alcohols, acids, esters, hydrocarbons, phenols, aldehydes, ketone and heterocycles, while alcohols (41.14%), acids (17.92%) and esters (29.37%) were dominant. Colony count number assay and rDNA amplicon-based high-throughput sequencing revealed 66 bacteria genera and 139 fungi during RSS fermentation, and Lactobacillus accounted for 94.81% in the first-round fermentation, while Pichia (49.71%), Dekkera (30.00%) and Saccharomyces (14.96%) were the dominant fungi in second-round fermentation. The Pearson correlation coefficient suggested that L. acetotolerans , Pichia , Dekkera and S accharomyces could contribute mainly to the special flavors of Kaili RSS. • Ripened Kaili Red Sour Soup (RSS) showed low nitrite risk. • Lactic acid, acetic acid and alanine contributed most to the specific taste of Kaili RSS. • Alcohols, acids and esters were dominant volatile compounds in Ripened Kaili RSS. • Kaili RSS experienced lacto-fermentation and lactobacillus -yeast co-fermentation. • L. acetotolerans , Pichia , Dekkera and Saccharomyces contributed mainly to Kaili RSS's flavor. [ABSTRACT FROM AUTHOR]