Systems and methods for secure enrollment of physical unclonable function devices include providing a device with an enrollment controller. The enrollment controller receives an enrollment request from an enrollment system and authenticates the request. If the request is authentic, the enrollment controller generates challenges in a pseudorandom order determined by a random seed that is shared with the enrollment system. The enrollment controller issues the challenges to interrogation circuitry coupled to a PUF array and records the responses. The responses are transmitted in encrypted form, and in the pseudorandom order, to the enrollment system. The responses are encrypted using a random number shared with the enrollment system. The enrollment system and the enrollment controller can independently generate the encryption key using the shared random number and/or other securely shared information.