To evaluate the value of MALDI-TOF MS for the risk monitoring of microorganism in strawberry and determine the hazards identification microorganism and their characteristics. Pseudomonas aeruginosa and Serratia marcescens were isolated from 50 strawberry samples by the selective culture method. The matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method was adopted to identify the microorganisms and SARAMIS Premium software was used to analyze and modify the protein mass spectrum of the two microorganisms by weighting. The results showed that the detection rate of Pseudomonas aeruginosa in strawberry was 18% and that of Serratia marcesmese was 20%. About 12 characteristic peaks of Pseudomonas aeruginosa isolated from strawberry completely fitted, and cluster analysis of identified peaks indicated that there were significant differences between the isolated Pseudomonas aeruginosa. There were 8 completely fitted characteristic peaks of Serratia marcesmese isolated from strawberry, and weighted clustering results of the identified peaks confirmed that the two isolated Serratia marcescens were homologous. The MALDI-TOF MS method can identify the specific characteristics of the protein isolated from strawberries and complicated microbial environment of agricultural products.