The invention discloses a physical isolation type USB port protection system and method. The protection system comprises a central processor, an upper computer connected with multiple user computers, an external memory used for storing user names and passwords and multiple relays used for controlling on-off of multiple USB ports in the user computers according to switching signals of the central processor. The upper computer is connected with the central processor through serial communication, and the control ends of the relays and the output end of the external memory are electrically connected with corresponding ports of the central processor. The protection method includes the steps that according to the user names, the central processor divides users into three levels, namely, common users, senior users and secret commissioners; the users input the user names and the passwords into the user computers, and the central processor controls login of the users according to the user names and the passwords; the central processor determines the levels of the users according to the user names and determines USB port use permission of the users. The protection method guarantees controllability of data in a computer.