In recent years, unmanned aerial vehicle (UAV) formation flight has become an effective strategy for urban air mobility (UAM). However, close rotor separation during formation flight leads to complex aerodynamic interference between rotors, significantly affecting UAV flight performance and operational safety. This study systematically examines the effects of axial and lateral rotor separation on the rotor’s thrust performance through wind tunnel experiments. The tests simulate horizontal, vertical, and hovering states by generating relative airflow in the wind tunnel, focusing primarily on the thrust coefficient changes of the bottom rotor at various separations. The results are compared with a single rotor operating under the same conditions without wake interference. Additionally, computational fluid dynamics (CFD) simulations using the Fluent software were conducted to investigate the effect of wake interactions by analyzing the velocity flow field between the two rotors in different separations. Both the experimental and simulation results demonstrate that rotor aerodynamic performance is notably influenced by wake interactions. Under hovering and vertical states, substantial aerodynamic interference occurs in the region directly beneath the top rotor, within 1D ≤ Z ≤ 3D. This interference gradually diminishes as the rotor separation increases. Additionally, the thrust coefficient of the bottom rotor decreases with increasing flight speed due to the wake, and at higher flight speeds, the wake tends to contract. When the lateral separation is X = 0D, the mid-sectional flow field of the two rotors exhibits symmetry; however, with lateral separation, the symmetry of the bottom rotor’s wake velocity field is disrupted. During the horizontal flight, the rotor wake tilts backward due to the relative airflow, and the extent of this influence is governed by both rotor rotational speed and flight velocity. Therefore, when UAVs operate in formation, it is crucial to account for these factors affecting aerodynamic performance, and rotor separation must be optimized to enhance flight safety and efficiency.