In order to optimize the distribution route of fresh agricultural products and reduce the distribution cost, the optimal route selection model of fresh agricultural products transportation based on bee colony algorithm is constructed. After establishing the transportation road network model of fresh agricultural products and analyzing the transportation road information, the transportation road network zoning is realized by the dynamic zoning method of transportation road network based on spectral clustering algorithm, taking the unblocked area and congested area as the zoning purpose. Based on the traffic zoning information, the transportation route optimization model of fresh agricultural products with time window is constructed. Solved by bee colony algorithm to obtain the distribution route of fresh agricultural products with the lowest distribution cost and the highest customer satisfaction. The experimental results show that the model can choose the fresh agricultural products distribution route with the lowest distribution cost and the highest customer satisfaction under the two working conditions of smooth traffic and congestion.