Purpose To generate microplasmin (lPlm) using recombinant microplasminogen (lPlg) and recombinant tissue plasminogen activator (rt-PA) before intravitreous injection and to investigate the efficacy of lPlm in inducing posterior vitreous detachment (PVD). Methods Forty-eight female or male New Zealand white rabbits were randomized into three groups. Recombinant human lPlg was incubated with rt-PA with a 200:1 molar ratio at 371C for 40 min. The right eyes of groups 1, 2, and 3, were injected with 0.5, 1.0, and 1.5 U lPlm in 0.1 ml respectively, and 0.1 ml balanced salt solution (BSS) was injected into the left eye as controls. Scanning electron microscopy (SEM), gross specimen examination, B-ultrasonography and optical coherence tomography (OCT) were performed to detect vitreoretinal interface. Results Over eighty percent of recombinant human lPlg could be activated to active lPlm by rt-PA after 40 min incubation. Complete PVD was found at vitreous posterior pole of lPlm-treated eyes without morphological change of retina. Complete PVD of 25, 75, and 87.5% rabbit eyes was induced by 0.5, 1.0 and 1.5 U recombinant lPlm respectively on day 1. The remnants of vitreous cortex at the posterior pole were dependent on the concentration of lPlm. Among the four approaches for detecting PVD, SEM, gross specimen examination, and B-ultrasonography were more effective methods than OCT. Conclusion Intravitreous injection of 1.5 U lPlm can effectively induce complete PVD in rabbit eyes on day 1 without morphological change of retina.