With the popularization of green concepts and the rapid growth of cold chain logistics demand, developing efficient cold chain technology is a focus. Currently, most research is concentrated on simulating the cooling performance of cold storage boxes under constant temperature conditions, whereas experimental investigation of cold storage boxes under dynamic temperature conditions remains insufficiently explored. In this paper, the impact of phase change material (PCM) packaging layout on the cooling performance of the cold storage box, internal temperature distribution, and practical application effects was studied. The variation of temperature inside the box under different numbers and placement positions of PCM packages was analyzed through experiments, and its applicability was verified under dynamic ambient temperature conditions. The results indicate that the temperature increase inside the box was reduced with an increased number of phase change packages, while top-mounted packages were found to effectively reduce cover heat leakage and significantly extend the cooling time. During the 35 h transportation process from Shanghai to Beijing, in the best case studied, the temperature inside the box could be maintained below 7.7°C, and the temperature increase rate was observed to vary synchronously with ambient temperature fluctuations. In addition, the thermal retention test demonstrated that the cold storage box could maintain thermal stability for up to 47.3 hours under extreme cold conditions in Harbin. Generally, this study provided a theoretical basis for optimizing cold chain transportation equipment.