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In the actual cultivation process, blind fertilizer application was widespread, resulting in a serious decline in the yield of Pseudostellaria heterophylla. We used the 3414 fertilizer experiment design to study the effects of combined Boron (B), Molybdenum (Mo), and Copper (Cu) on the growth indexes, diurnal changes of photosynthesis, and rapid fluorescence induction dynamics in P. heterophylla. Our results show that the optimal combination of B, Mo, and Cu simultaneously promoted the growth of underground and aboveground parts, and significantly improved the quality of single root tuber and yield per unit area. The best combination was treatment 9 (T9 = B, 1 g/L; Mo, 0.08 g/L; Cu, 0.05 g/L), and resulted in a 35.1% increase in yield per unit area compared with the control group (T1). Although the optimal combined application of microfertilizers did not change the bimodal trend of diurnal variation of photosynthesis, it effectively increased the daily average, peak, and valley values of the photosynthetic rate by alleviating the nonstomatal limitation and the photosynthetic midday depression. Pseudostellaria heterophylla leaves showed greater photochemical activity and less photoinhibition of photosystem II in T9. Major effects were that it helped protect the activity of the oxygen-evolving complex to reduce the oxidative damage of chloroplasts and prevent the dissociation of thylakoid. The microfertilizer application also enhanced the electron receiving ability of the QB and plastoquinone (PQ) electronic pools, thereby increasing the ability of electron transfer from QA to QB. The number of reaction centers per unit area was promoted notably by the fertilization treatment.