The invention provides an immortalized mouse microglial cell line B6Mi1. A preservation number of the immortalized mouse microglial cell line B6Mi1 in CCTCC (China Center for Type Culture Collection)is CCTCC NO:C2019263, and a preservation date is November 6, 2019. A creation method of the mouse microglial cell line includes steps: a, primary mouse microglial cell separation and culture; b, primary mouse microglial cell purification and cloning. Mouse microglial cells are spontaneous immortalized microglial cells and are in IBA1 positive, CD45<+>CD11b<+>. B6Mi1 cells are highly proliferative,cell cycle analysis shows that more than half of the cells are in an S stage and a G2M stage, and cell doubling time is 14 hours. By LPS processing, expression of inflammatory factors including TNFalpha, IL-1beta, IL-6, iNOS and the like can be remarkably increased. According to the results, the B6Mi1 cells belong to the mouse microglial cell line with microglia cell phenotype and biological characteristics, and the cell line can be used for researching biological characteristics of the microglial cell and neuroinflammation and screening and development of related medicines.