Given the overlap in time-frequency domain of code division multiple access (CDMA) multi-user signals, we propose a novel estimation method for time difference of arrival (TDOA) and frequency difference of arrival (FDOA). Based on the acquiring and dispreading of spread spectrum signals, with short-term signal samples and limited computation, the method adopts only two iterations of time-frequency searching to separate signals and estimate time frequency difference, and then uses the time domain and frequency domain interpolation to further improve estimation accuracy. Simulation results show that the presented algorithm can effectively improve the estimation accuracy of time-frequency difference of CDMA signals and lower the computational complexity in comparison with the cross ambiguity function correlation method.