Abstract: Objective:In the paper, the process conditions of adsorption and separation of gentamicin by macroporous weakly acidic resin Amberlite FPC3500 were studied.Methods:Through static or dynamic adsorption and dynamic desorption experiments of resin, the preferable technical conditions of reclaiming gentamicin by FPC3500 resin were studied by using single factor experiment and orthogonal experiment, taking the adsorption rate, desorption rate and recovery ratio as the investigation index.Results:the optimal condition of static adsorption was at pH7.0 with 1h, room temperature, rotating rate of 140r /min.The recovery ratio was up to 94.8% at pH7.6, concentration of sampling 5.64g /L, flow rate of sampling 0.6mL /min, concentration of ammonia 7%.Conclusion:Macroporous weakly acidic resin was suitable for the separation and purification of gentamicin, adsorption capability was better than the commonly-used strong acid resin of gel type in our country.