A multi-port power supply device and an operation method thereof are provided. The multi-port power supply device includes a power converter, a power switch, a current detection circuit, a voltage detection circuit, a control circuit, and multiple USB ports. The power converter supplies power to a USB port via a current path. The control circuit determines whether the USB port is connected to a USB device according to an actual voltage of the current path. When the USB port is not connected to the USB device, the control circuit turns off the current path. When the USB port is connected to the USB device, after a part of a power of other USB ports is dynamically transferred to the USB port, the control circuit determines whether to turn on the current path according to an actual current of the current path.