In this paper, a two-step resin adsorption technology was investigated for spinosad production and separation as follows: the first step resin addition into the fermentor at early cultivation period to decrease the timely product concentration in the broth; the second step of resin addition was used after fermentation to adsorb and extract the spinosad. Based on this, a two-step macroporous resin adsorption-membrane separation process for spinosad fermentation, separation, and purification was established. Spinosad concentration in 5-L fermentor increased by 14.45 % after adding 50 g/L macroporous at the beginning of fermentation. The established two-step macroporous resin adsorption-membrane separation process got the 95.43 % purity and 87 % yield for spinosad, which were both higher than that of the conventional crystallization of spinosad from aqueous phase that were 93.23 and 79.15 % separately. The two-step macroporous resin adsorption method has not only carried out the coupling of spinosad fermentation and separation but also increased spinosad productivity. In addition, the two-step macroporous resin adsorption-membrane separation process performs better in spinosad yield and purity.