In order to improve the shale oil production rate and save fracturing costs, based on dynamic production data, a production-oriented optimization method for fracture spacing of multi-stage fractured horizontal wells is proposed in this study. First, M. Brown et al.’s trilinear seepage flow models and their pressure and flow rate solutions are applied. Second, deconvolution theory is introduced to normalize the production data. The data of variable pressure and variable flow rate are, respectively, transformed into the pressure data under unit flow rate and the flow rate data under unit production pressure drop; and the influence of data error is eliminated. Two kinds of typical curve of the normalized data are analyzed using the pressure and flow rate solutions of M. Brown et al.’s models. The two fitting methods constrain each other. Thus, reservoir and fracture parameters are interpretated. A practical model has been established to more accurately describe the seepage flow behavior in shale oil reservoirs. Third, using Duhamel’s principle and the rate solution, the daily and cumulative production rate under any variable production pressure can be obtained. The productivity can be more accurately predicted. Finally, the analysis method is applied to analyze the actual dynamic production data. The fracture spacing of a shale oil producing well in an actual block is optimized from the aspects of production life, cumulative production, economic benefits and other influencing factors, and some significant conclusions are obtained. The research results show that with the goal of maximum cumulative production, the optimal fracture spacing is 5.5 m for 5 years and 11.4 m for 10 years. All in all, the fracture spacing optimization and design theory of multi-stage fractured horizontal wells is enriched.