To evaluate whether matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method could be used to identify Clostridium perfringens rapidly, 51 C. perfringens isolated strains were identified by MALDI-TOF MS method and compared with results from VITEK 2 COMPACT, 16S rRNA gene sequencing and iron-milk presumptive test. All the isolated strains were identified as C. perfringens by MALDI-TOF MS method and the Log score were all above 2.300, indicating accurate species identification. The identification results from 16S rRNA sequencing were the same as above. Two C. perfringens strains were identified as “Unidentified Organism” with VITEK 2 COMPACT. Four C. perfringens strains got negative results in iron-milk presumptive test. These results indicated that both iron-milk presumptive test and VITEK 2 COMPACT could give false negative results in C. perfringens identification. Compared to 16S rRNA sequencing, iron-milk presumptive test and VITEK 2 COMPACT, MALDI-TOF MS method was more accurate, rapid and lower cost. It was more suitable for C. perfringens identification.