In order to ensure the safe operation of urban logistics UAVs in the free flight scenario, the required separation for UAVs should be equipped. Gas model and position error probability model were adapted, and method for determining the free flight separation of UAVs covering conflict detection and resolution mechanism was proposed. Firstly, gas model was used to evaluate the conflict frequency of UAVs, and the collision probability when the conflict occurs were calculated by considering air traffic characteristics, conflict detection and resolution mechanism, route structure as well as air traffic service capability. Total collision probability was obtained by synthesizing the two values. Finally, the safety target level is benchmarked and the required separation for free flight of urban logistics UAV was calculated. Using this method, taking the free flight of UAV in a certain operating space as an example, the required separation was calculated. The research results provide a feasible method for the formulation of urban logistics UAV separation minima. The method can also be used to guide the CDR response mechanism to reduce the collision probability after the separation is breached.