Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry viaAedes albopictusbites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the geneAALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) ofAe. albopictusand studied the salivary protein-mediated enhancement of DTMUV infection inAe. albopictussalivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa inAe. albopictusis currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein inAe. albopictussaliva that could serve as a target for controlling DTMUV replication in mosquito vectors.