Alphaviruses mainly include old-world alphaviruses that cause arthritis and new-world alphaviruses that cause fatal encephalitis, which can lead to serious diseases in humans. In order to establish a safe and convenient tool to study the entry mechanism of alphaviruses, this study established pseudovirus particle system-based on murine leukemia virus (MLV) and virus replicon particle (VRP) system-based on Sindbis viurs (SINV). Using these pseudoviruses and VRPs, the effects of knockout of heparan sulfate biosynthesis related gene B3 gat3 (beta-1, 3-glucuronyltransferase 3) and arthritogenic alphavirus related entry receptor gene Mxra8 (matrix remodeling associated 8) on cell entry of different alphavirus members were verified. Therefore, the MLV-based pseudovirus and SINV-based VRP systems in this study provide a safe and reliable experimental tool for the research on the entry mechanism of alphavirus members and the development of antiviral strategy.