Objective To compare the differences in virulence-related factor aspartate protease, biofilm formation, and gene expression among clinical isolates of Candida parapsilosis (C. parapsilosis). Methods Gene sequencing and microsatellite typing (MT) method were adopted to identify C. parapsilosis isolated from patients with clinical fungal infection. The production of secreted aspartate protease and biofilm formation ability of each strain were detected, and the expression of biofilm formation related-genes BCR1, EFG1, and HWP1, as well as aspartate protease virulence genes SAPP1, SAPP2, SAPP3 were compared among the strains. Results A total of 8 clinically isolated C. parapsilosis strains were collected, all of which were identified as genotype Ⅰ. Based on microsatellite typing results, 8 clinical strains were divided into 4 microsatellite types. G1, G2, and G3 strains isolated from the urine, peripherally inserted central catheters (PICC), and blood of patient A were of different subtypes. J1, J2, J3, J4, and J5 strains were of the same type, and isolated from blood specimens of patient B at different periods. All 8 clinical strains could form biofilm, and their biofilm formation ability was higher than that of the standard strain of C. parapsilosis (ATCC 22019). G1, G3 and J5 strains had strong biofilm formation ability, J1, J2, J3, and J4 strains had moderate biofilm formation ability, and G2 strain had weak biofilm formation ability. All of the eight clinical isolates secreted aspartate protease, and their in vitro expression levels of the enzyme were higher than that of the standard strain (ATCC 22019). G3, G1, and G2 strains showed low, moderate, and high in vitro enzyme expression respectively, with statistical differences (all P<0.05). Enzyme expressed moderately in J1 and J5 strains, and highly in J2, J3, and J4 strains. Difference between moderate and high expressions was statistically significant (P<0.05). The expression levels of biofilm formation genes BCR1, EFG1, and HWP1 in various strains isolated from patients A and B increased. In strains isolated from patient A, the expression level of EFG1 gene in G1 strain was higher than that in G2 strain (P<0.05). There was no statistically significant difference in BCR1, EFG1, and HWP1 gene expression levels among strains isolated from patient B. The expression levels of aspartate protein genes (SAPP1, SAPP2, and SAPP3) in various strains isolated from patients A and B increased. The expression levels of SAPP1 and SAPP2 in strain G1 were higher than those in G2 and G3 (both P<0.05). There was no statistically significant difference in the expression levels of SAPP1, SAPP2, and SAPP3 genes in strains from patient B. Conclusion Clinical isolates of C. parapsilosis have higher biofilm formation and aspartate protease production abilities than standard strain. The expression of virulence factors varies among strains isolated from different specimens, while there is no significant difference in the expression of virulence factors among strains isolated at different periods. Patients may have been infected with different MT types of C. parapsilosis in multiple sites during the same period.