Abstract: In multi-user differential frequency hopping (DFH) systems, different frequency transition functions are assigned to each user. To mitigate multipath interference and multi-user interference in asynchronous uplink communication of underwater acoustic multi-user DFH systems, a time-varying frequency transition function generation algorithm with joint optimization of the two types of interference is proposed. The algorithm suppresses multipath interference within the minimum free distance by constraining the trace of consecutive products of single-user frequency transition matrices, while mitigating multi-user interference across successive adjacent hopping periods by constraining the sum of Hadmard products of multi-user frequency transition matrices. Experimental results from Qiandao Lake demonstrate that the proposed algorithm, with a 1/4 code rate, enables error-free communication for each user at a rate of 62.5 bit/s within a 2.5 km range in the 3−5 kHz frequency band, even when four users are asynchronously transmitting from different locations and distances. The signal-to-noise ratio (SNR) threshold for this performance is −5 dB. Moreover, the bit error rate is better than that of conventional frequency hopping systems and time-invariant DFH systems.