With the improvement of people’s living standards and the widening of circulation channels, the demand for fresh agricultural products continues to increase. The increase in demand will lead to an increase in delivery vehicles, costs, and carbon emissions, among which the increase in carbon emissions will aggravate pollution and is not conducive to sustainable development. Therefore, it is very important to balance economic and environmental benefits in the distribution of fresh agricultural products. Based on the analysis of the distribution characteristics of fresh agricultural products, this paper studies the optimization of the cold chain distribution route of fresh agricultural products considering carbon emission. Firstly, the cold chain distribution route planning of fresh agricultural products was investigated and analyzed by the interview method, and the basis for establishing the model objective and constraint conditions was obtained. Then, taking the minimum total cost including carbon emission cost as the optimization goal, the cold chain distribution route optimization model for mixed vehicle types is established considering electric refrigerated vehicles, gasoline refrigerated vehicles, and so on. Genetic algorithm was used to solve the model, and MATLAB2018b was used to substitute specific case data for simulation analysis. The analysis results show that increasing the consideration of carbon emission and mixed vehicle types in the distribution route of fresh agricultural products can not only reduce the distribution cost but also reduce the carbon emission. To some extent, the research content of this paper can provide a reference for enterprises in planning cold chain distribution routes of fresh agricultural products.