The work aims to propose an ambulance layout optimization design method based on the hand movement trajectory observation method and optimize the cabin layout by analyzing the emergency workers' rescue postures and behavioral trajectories, thus improving the work efficiency of emergency workers. The emergency workers' behaviors were observed by the hand movement trajectory observation method. Combined with hierarchical task analysis, the work tasks were classified and the layout zoning analysis and mapping analysis were used to link user behaviors with cabin layout and interactive components. SPSS tools were employed for correlation analysis of trajectory data. Hand movement trajectory data for classic emergency tasks under different ambulance cabin layouts were obtained. Based on data analysis, optimization strategies for ambulance cabin layout were derived, and innovative design schemes for ambulance cabins were developed. Different ambulance cabin layouts lead to significant differences in task trajectories, and there is a high correlation between layout data indicators and trajectory data indicators. Analyzing layout data can yield clear optimization strategies for layout design to improve trajectory data, optimize emergency processes, and prove that the hand movement trajectory observation method can serve as a design method for optimizing cabin space layout.