The invention discloses a separation and purification technology for extracting gentiopicrin from radix gentianae macrophyllae, and belongs to the technical field of extraction, separation and purification of the gentiopicrin. The separation and purification technology comprises the following steps: firstly, selecting an extraction solvent and controlling extraction temperature; carrying out orthogonal test on different extraction solvents and different extraction temperatures to determine that the best extraction solvent is purified water; by using macroporous resin, carrying out initial separation and purification on an extracting solution to determine which resin is selected; when the macroporous resin is used for crude separation, selecting the type of eluent and determining the concentration and the use amount of the eluent, as well as a starting point and an end point of the eluent collection; carrying out further purification and dehydration on eluent by using D101 macroporous resin and then recrystallizing the obtained eluent. According to the separation and purification technology disclosed by the invention, high purity gentiopicrin is obtained by separation and purification by utilizing the separation ability of the D101 macroporous resin and different solubilities of effective ingredients in the solvent, and the extraction rate, the separation purity and the overallyield of the gentiopicrin are improved.