To enhance the biofortification of B vitamins in food, golden oyster mushrooms (Pleurotus citrinopileatus) were used as the experimental material. Amino acid precursors were added during mushroom cultivation to increase B-vitamin content. Additionally, a method for the simultaneous quantification of multiple B vitamins in golden oyster mushrooms was developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Single-factor fortification experiments showed that optimal precursor concentrations of L-aspartic acid (100 mg•kg-1), β-alanine (500 mg•kg-1), and L-glutamic acid (10 mg•kg-1) increased the contents of vitamins B3, B5, and B9 to 88.78, 80.92, and 85.78 mg/100 g, respectively, corresponding to 1.21-, 1.64-, and 1.09-fold increases compared with those in the control group. A synergistic extraction system combining formic acid and protease was identified as the optimal extraction method. The composition and content of B vitamins in golden oyster mushrooms subjected to combined amino acid fortification were further analyzed using HPLC-MS/MS. The results showed that coaddition of the three amino acids produces a stronger fortification effect than single-factor treatments, resulting in a total B-vitamin content of 309.31 mg/100 g, which was 2.48-fold higher than that of the control group. In conclusion, amino acid co-fortification during cultivation effectively enhanced B-vitamin accumulation in golden oyster mushrooms, and the formic acid + protease method enabled efficient extraction. These findings provide theoretical support and technical guidance for B-vitamin biofortification in edible fungi.