Abstract: Gamma spectrometry analysis of uranium enrichment is an important technical tool for international nuclear safeguards verification. As a kind of special nuclear materials, ²³⁵U is an important verification object of nuclear safeguards verification. In addition, the determination of uranium enrichment in samples is a key measurement for product process control, process measurement and waste characterization in uranium enrichment production facilities, uranium conversion facilities, fuel manufacturing plants and reprocessing plants, as well as a key technical means for tracking illicit trafficking in nuclear materials and responding to terrorist threats and attacks in homeland security activities. Simulated annealing algorithm is aiming to simulate the solid annealing process, and iteratively solve the solution problem by continuously reducing the annealing temperature and combining with the Metropolis criterion. The result has been proved to converge to the global optimal solution according to probability 1, which is one of the common solutions for solving optimization problems. In the article, a uranium enrichment calculation method based on simulated annealing algorithm is proposed for gamma spectrometry analysis. A corresponding mathematical model is established for the practical problem of enrichment calculation, the generation and iterative process of algorithmic solutions are improved by using the sampling theory and the forbidden searching strategy, and the iterative solution is combined with the nonlinear least-squares fitting for the enrichment of the samples, which realizes the relative efficiency of the curve fitting and the calculation of the enrichment degree within the energy range of 143 keV to 1001 keV. The performance test of the algorithm and the results of sample analysis show that the uranium enrichment calculation method based on the improved simulated annealing algorithm has the ability to escape the local optimal problem in uranium enrichment calculation, and can be applied to the enrichment calculation of different uranium samples, the relative deviation of the results of the enrichment analysis over all measured samples is within ±5%.