In order to realize the rapid determination of ractopamine content in pork, quantitative determination model of ractopamine content in pork was established by using three-dimensional synchronous fluorescence spectrum coupled with alternating penalty trilinear decomposition (APTLD). Firstly, the generation mechanism of the fluorescence spectrum for ractopamine and three-dimensional synchronous fluorescence spectrum for samples were analyzed. Secondly, concentration quenching phenomenon of fluorescence of ractopamine in pork extract was investigated. Thirdly, the number of components for three linear decomposition of APTLD was set as 2 by using the core consistency diagnostic method, and the calibration curve of the relative fluorescence intensity of ractopamine between pork extract and the training sample was established for the correction of relative fluorescence intensity of prediction samples. Finally, three-dimensional synchronous fluorescence spectrum combined with APTLD was used to build the prediction model of ractopamine content in pork. The experimental results showed that the method adopted in the paper could better solve the problem of serious synchronous fluorescence spectrum overlapping between ractopamine in pork samples and backgrounds, and leave out some trivial process of chemical separation for the identification of ractopamine in pork. The determination coefficient (R2) and the root mean squared error of prediction (RMSEP) for the model proposed in this paper were 0.986 3 and 0.496 6 mg·L-1, respectively. The method in this paper has achieved the goal of rapid quantitative detection of ractopamine content in pork.