A fabrication method of micro-injection mold cavity for a cell culture device was explored in this paper. Based on the structural characteristics of the micro-injection mold cavity and overlay UV-LIGA technology, a microstructure using appropriate alloy steel as the substrate was fabricated through SU-8 photoresist optical lithography and micro-electroforming technology respectively twice. Then, a micro exhaust duct was etched on an electroforming deposit through mask etching technology. Meanwhile, the swelling problem of SU-8 thick photoresist, poor surface evenness through spin-coating and SU-8 photoresist removal in the experiment process were analyzed, and a method of designing and fabricating a 20 μm wide closed isolation strip around the mask graphics was presented to reduce the volume of SU-8 thick photoresist around the graphics and to improve the swelling deformation of plating mold. By which, the dimension error of the electroforming deposit is from 35 μm down to 10 μm and 300 μm high micro-square-cylinder side wall is steep. The introduction of closed isolation strip improves the dimensional and shape precisions of nickel electroforming deposit effectively and the uses of scraping photoresist and oleum techniques eliminates the edge bead effect and removes SU-8 photoresist completely. As a result, the micro-injection mold cavity with good quality and the high bonding strength between substrate and nickel electroforming deposit is obtained successfully.