The present invention provides a method for detection of malicious RFID (Radio Frequency Identification) reader intrusion. The method comprises the steps of: obtaining throughput rate relative deviation and a received signal intensity of a valid RFID reader in real time; obtaining a throughput rate relative deviation predicted value through the received signal intensity and a fit curve; and determining whether there is malicious RFID reader intrusion at a current moment or not in real time according to a relation of the throughput rate relative deviation, the throughput rate relative deviationpredicted value and a confidence limit of the valid RFID reader at the current moment. The present invention further provides a device for detection of malicious RFID reader intrusion. The device comprises a first obtaining module, a second obtaining module, a third obtaining module and a detection module. The method and the device provided by the invention employ hardware of a RFID system itselfto achieve accurate detection of the malicious RFID reader intrusion in a condition without an influence on normal work of a RFID device and improvement of a detection cost.