Objective To investigate the antibacterial properties of R.lnPGRP-SC1a in Rhipicephalus linnaei, and to provide a reference for the functional analysis of peptidoglycan recognition proteins (PGRPs) within the context of tick innate immunity. Methods Specific primers were meticulously designed based on the coding region sequence, followed by the extraction of tick RNA of adult ticks and its reverse transcription into cDNA. The R.lnPGRP-SC1a gene fragment was subsequently amplified via PCR and then ligated into the plasmid pET32a+, thereby constructing the recombinant expression vector pET32a+-R.lnPGRP-SC1a. This expression vector was then transferred into E.coli BL21 (DE3) competent cells and induced with an IPTG concentration of 0.2 mmol/L at low temperature to enhance protein expression in the supernatant, thereby obtaining a soluble protein with stronger activity. Subsequently, the inhibitory effect of the supernatant protein against two common pathogenic bacteria, E.coli and S.aureus, was assessed using the agar diffusion method. Results The amplified gene fragment was 627 bp in length, and the prokaryotic expression vector pET32a+-R.lnPGRP-SC1a was successfully constructed. Low-temperature induction showed that the recombinant protein was soluble protein, with an approximate molecular weight of 23.63 kD. Antibacterial activity results indicated that, at the same concentration, R.lnPGRP-SC1a exhibited no inhibitory effect on E.coli but demonstrated significant inhibition against S.aureus. Specifically, antibacterial activity became evident at a concentration threshold of 5 mg/mL and increased with the protein concentration. Compared with the inhibitory effects of kanamycin at various concentrations, the inhibitory effect of R.lnPGRP-SC1a at 7 mg/mL was comparable to that of kanamycin at 1 mg/mL. Furthermore, the onset of R.lnPGRP-SC1a inhibitory effect against S.aureus was 2 hours, and the effect lasted for 48 hours. Conclusions This study successfully constructed the R.lnPGRP-SC1a expression vector, its expressed product exhibited persistent activity against Gram-positive bacteria. Thereby, this provides a potential possibility for the development of bioactive bacteriostatic agents.