Pattern recognition receptors (PRRs) are biosensor proteins that bind to non-self pathogen associated molecular patterns (PAMPs). β-1,3-glucan recognition proteins (βGRPs) play an essential role in immune recognition and signaling pathway of insect innate immunity. Here, we report the cloning and characterization of cDNA of OfβGRP3 from Ostrinia furnacalis larvae. The OfβGRP3 contains 1455 bp open reading frame, encoding a predicted 484 amino acid residue protein. In hemocytes, the expression levels of OfβGRP3 in Escherichia coli-challenged group were higher than those of Bacillus subtilis-challenged group at 2, 4, 8, 10 and 12 h post injection (HPI). In fat body, OfβGRP3 expression in both B. subtilis and E. coli-challenged group was significantly higher than that in untreated group from 4 to 10 HPI, and then the expression continuously dropped from 12 to 36 HPI. The OfβGRP3 expression in laminarin-injected group was higher than that in lipopolysaccharides (LPS)-injected group in various test tissues from 4 to 24 HPI. The LT50 of E. coli-infected OfβGRP3-RNAi larvae (1.0 days) was significantly lower compared with that of E. coli infected wild-type larvae (3.0 days) (p < 0.01). Only 10.2% Sephadex G50 beads (degree 3) were completely melanized in the larvae inoculated with OfβGRP3 dsRNA, as compared to 48.8% in control larvae (p < 0.01). A notable reduction in the PO activity and IEARase activity in hemolymph was also detected in the OfβGRP3 knockdown larvae. Our study demonstrates that OfβGRP3 is one of PRR members involved the PPO-activating system in O. furnacalis larvae.