The present invention relates to a method for on-line detection of the internal resistance of a storage battery by performing low-direct-current discharge twice. The low current discharge is performed twice on the storage battery in a load state, and two discharge loops are composed of switches and two precision resistors having different resistances. The on-line detection method is provided based on the direct current high current discharge method, a sampling holder is employed in the process of the voltage acquisition to perform sampling holding of voltages acquired in the two-time discharge, then a required voltage difference in the two-time discharge is obtained through differential amplification, and the internal resistance of the storage battery is calculated according to the voltage difference in the two-time sampling. According to the on-line detection method provided by the invention, the problems of the damage of a high current discharge method to a storage battery and the defect of an inappropriate on-line detection may be overcome, voltage difference in the two-time discharge obtained through sampling holding and differential amplification may improve the precision of data acquisition, and the error is reduced, therefore, the exact value of the internal resistance of the storage battery is obtained and the accurate detection of the internal resistance of the storage battery in the load state is realized online.