Serine proteases are involved in regulation of innate immune responses, such as haemolymph coagulation, melanization reaction and antimicrobial peptide synthesis. Although several serine proteases have been characterized in Anopheles gambiae (A. gambiae), few were cloned from other malaria vectors. In this study, we identified three cDNA fragments of serine proteases (AdSp1, AdSp2 and AdSp3) from haemocytes of an oriental malaria vector, Anopheles dirus (A. dirus), by cloning of fragments amplified with degenerate primers into the T-vector. RT-PCR analysis demonstrated that both AdSp1 and AdSp3 genes were also expressed in salivary gland. Basic local alignment search tool (BLAST) search found that both AdSp1 and AdSp3 were highly similar in sequence to A. gambiae Sp14A and Sp14D2, insects prophenoloxidase activating enzyme (PPAE) and Drosophila protease easter. Semi-quantitative RT-PCR indicated the transcription level of both AdSp1 and AdSp3 in haemocytes of A. dirus infected with Plasmodium yoelii (P. yoelii) was significant higher than that fed on 5% glucose or normal mouse blood at 7 days after the infectious meal (p<0.05), when P. yoelii oocysts began to be melanized by A. dirus. Our results indicated that both AdSp1 and AdSp3 might play an important role during melanotic encapsulation of P. yoelii by A. dirus.