The invention belongs to the technical field of biological fermentation and particularly discloses a method for increasing the yield of bacillus subtilis vitamin B2. The method includes the followingsteps of 1, adding an alkaline liquid into a fermented liquid quickly under a forced stirring condition without light at the temperature lower than 30 DEG C and adjusting the pH value of the fermentedliquid to be 11-12; 2, adding a flocculant into the alkaline-treated fermented liquid for flocculation; 3, sequentially adopting centrifugal separation and membrane filtering for conducting solid-liquid separation on the fermented liquid after flocculation; 4, acidizing a clear liquid obtained by solid-liquid separation in step 3, adding seed crystals into the clear liquid for crystallization toobtain coarse crystals, conducting crystallization several times, and making the obtained crystals subjected to washing and drying to obtain a finished product. By optimizing extraction procedures ofthe fermented liquid, the yield of the vitamin B2 is increased.