A signal calibration method, a memory storage device, and a memory control circuit unit are provided. The signal calibration method includes: generating a clock signal and a data strobe signal according to an internal clock signal; respectively transmitting the clock signal and the data strobe signal to a target volatile memory module among multiple volatile memory modules through a first signal path and a second signal path; obtaining a shift value between the data strobe signal and the clock signal at the target volatile memory module; and storing an initial delay setting of the data strobe signal according to delay information of the data strobe signal in response to the shift value being greater than a threshold value.