Neutral beam injection (NBI) is one of the most effective means of plasma heating and current driving. There are three basic processes in neutral beam injector: generation of ion beam,neutralization of ion beam and transmission of neutral beam, among which neutralization of ion beam is the key link. The gas target thickness in neutralizer directly affects the neutralization process, and further affects neutral beam transmission efficiency.A new diagnostic technique for the determination of gas target thickness, based on the Doppler shift effect, is presented and applied to neutral beam injector operating on Experimental Advanced Superconducting Tokamak (EAST) NBI testbed. The basis of the method is the process of beam component evolution with gas target thickness.The gas target thickness is estimated by the value of Dalpha spectral line intensity. The method is applied to the EAST NBI of Institute of Plasma physics, Chinese Academy of Sciences (ASIPP). Gas target thickness in the range of 0.16~0.22×1016 cm-2 are measured close to the exit of neutralizer,and gas target thickness measured with this novel technique correlate very well with the extracted beam current. According to the law of conservation of mass, a rough calculation of gas target thickness is basically consistent with the calculation of the novel technique, which verifies its correctness. Experimental results show that spectrum method based on the Doppler shift effect can be applied to estimate neutralizer gas target thickness.