In order to investigate the influence of multi-strain fermentation to the flavor substances and sensory evaluation of soy sauce， there are four groups of experimental were designed： group Ⅰ （fermentation only with Aspergillus oryzae 3.042）， group Ⅱ （Aspergillus oryzae 3.042+Saccharomyces rouxii AS2.180）， group Ⅲ （Aspergillus oryzae 3.042+Saccharomyces rouxii AS2.180+Torulopsis bombicola huniang2.14）， group Ⅳ（Aspergillus oryzae3.042+Saccharomyces rouxii AS2.180+Torulopsis bombicola huniang2.14 +Lactobacillus huniang1.08）， and compared of the 4 groups after fermented 90 d. The result showed that： （1）The physicochemical indexes of ammoniacal nitrogen， reducing sugar and pH is similar， but the content of ammoniacal nitrogen in group Ⅳ is slightly higher than the other 3 groups. （2） Saccharomyces rouxii AS2.180 promotes the formation of higher alcohols and aromatic heterocyclic compounds. Torulopsis bombicola huniang2.14 promotes the formation of 4-EG and 4-VG that makes soy sauce had a special aroma. Lactobacillus huniang 1.08 makes the soy sauce tastes refreshing. And the difference of the types and content of the flavor substances is very significant of the 4 groups （P<0.01）. （3） Using suitable functions to describe the relationship between characteristic flavor substance content and the time of fermentation can get a mathematical relationship （such as： group Ⅰ y = -230.83+21.7 t - 0.136 t2 + 1.27×10-4 t3）， and can figure out the largest content of characteristic flavor substance of the 4 groups is： 648.96 μg/L （group Ⅰ： fermentation period is 92 d）， 852.90 μg/L （group Ⅱ： fermentation period is 98 d）， 982.51 μg/L（group Ⅲ： fermentation period is 99 d）， 1 897.55 μg/L （group Ⅳ： fermentation period is 102 d） respectively. Multiple fungus fermentation can increase the characteristic flavor substance content in soy sauce.