Pinctada fucata martensii (P. f. martensii) is one of the main pearl oysters cultured in artificial seawater in China. However, it is highly susceptible to pathogen infection under intensive cultivation near the coast. MicroRNAs (miRNAs), as an innovative and potent regulator of immune function, play a pivotal role in the immune response of pearl oysters to external stimuli and are a potent marker for the response of P. f. martensii to infection. This study identified two novel miRNAs, novel-13 and novel-44, from the whole transcriptome of the P. f. martensii hemocyte before and after infection with Vibrio parahaemolyticus. The dual luciferase results showed that novel-13 negatively regulated LAAO and novel-44 negatively regulated ILK. The activity of antioxidant-related enzymes increased significantly in the synthetic miRNA (novel-13 and novel-44) inhibitors and decreased significantly in the synthetic miRNA mimics. In the challenge experiment, injection with miRNA inhibitor increased the relative survival percentage by 10% compared with the control group. In conclusion, the overexpression of novel-13 and novel-44 can decrease the activity of immune and antioxidant-related enzymes, possibly affecting immune regulation in P. f. martensii by negatively regulating the LAAO and ILK target genes.