Abstract: Aim： To clone and over-express the gene encoding aminoglycoside (AG) phosphotransferase (APH) from clinical MRSA isolates in E.coli and to develop an assay method for the recombinant APH. Methods: The susceptibility of clinical MRSA isolates to AGs was tested by disk diffusion.A nucleic acid sequence encoding APH was amplified from the genomic DNA of an isolate and ligated to expression vector pET-28a，and then transformed into E.coli BL21 (DE3).After purification of the recombinant protein by affinity chromatography，the phosphorylation activity of the enzyme was determined by ESI-MS and disk diffusion. Results： All 6 clinical MRSA isolates were unsusceptible to AGs.After cloning and expression，the recombinant APH was purified to 90%. The in vitro activity assay indicated that the recombinant protein could inactivate kanamycin B in the assay mixture within 2 h. Conclusion： The recombinant APH showed excellent enzymatic activity.The assay method was simple and convenient，which may provide the basis of developing a screening model for APH inhibitors.