An accurate time-of-arrival (TOA) estimation method for isolated pulses positioning system is proposed in this paper. The method is based on a multi-level crossing timing (MCT) digitizer and least square (LS) criterion, namely LS-MCT method, in which TOA of the received signal is directly described as a parameterized combination of a set of MCT samples of the leading and trailing edges of the signal. The LS-MCT method performs a receiver training process, in which a GPS synchronized training pulse generator (TPG) is used to obtain training data and determine the parameters of the TOA combination. The LS method is then used to optimize the combination parameters with a minimization criterion. The proposed method is compared to the conventional TOA estimation methods such as leading edge level crossing discriminator (LCD), adaptive thresholding (ATH), and signal peak detection (PD) methods. Simulation results show that the proposed algorithm leads to lower sensitivity to signal-to-noise ratio (SNR) and attains better TOA estimation accuracy than available TOA methods.