In view of the consumer order demand of multi-variety, high frequency, small batch, high timeliness and high added value, the work aims to propose a whole cold chain joint pickup and distribution model based on road-rail transportation with the help of self-developed and designed multi-temperature refrigerated container, and conduct empirical research on it. With the objective of minimizing the total cost, a whole joint pickup and distribution optimization model of multi-temperature refrigerated container based on road-rail transportation was constructed and compared with the multi-temperature joint distribution model of whole mechanical refrigerated vehicles. The genetic algorithm was used to optimize the solution, and sensitivity analysis was carried out in terms of distribution time efficiency and high-speed railway transportation price. Compared with the multi-temperature joint distribution model of whole mechanical refrigerated vehicles, the total cost of whole cold chain joint pickup distribution of multi-temperature refrigerated container based on road-rail transportation was reduced by 38%, with the cost of refrigeration, carbon emissions and time penalties being particularly reduced. With the normal distribution time constraint as the reference, extending or shortening the time window, the time penalty costs was reduced by 61% to 83%. Compared with the railway freight prices, under the condition that the high-speed rail freight prices would not rise by no more than 220%, the whole cold chain joint pickup and distribution model of multi-temperature refrigerated container based on road-rail transportation was more advantageous in terms of both economic and social benefits. The whole cold chain joint pickup and distribution model of multi-temperature refrigerated container based on road-rail transportation can reduce costs and increase efficiency, save energy, reduce emissions for enterprises, and provide a new model of operation for transport entities to integrate idle transport capacity.