The invention discloses a differential detection method based on a distributed optical fiber, application thereof, and a protection system. According to the differential detection method based on the distributed optical fiber, before detecting an intrusion behavior, a detection optical fiber needs to be fixed at a signal detection sensitive point of a monitoring region, a reference optical fiber needs to be fixed at a signal detection insensitive point of the monitoring region, a monitoring host judges whether the detection optical fiber and the reference optical fiber generate vibration signals simultaneously, and the monitoring host determines that an intrusion behavior happens if the reference optical fiber does not generate a vibration signal while the detection optical fiber generates a vibration signal. According to the differential detection method, the intrusion behavior is detected by adopting the difference method, the detection precision of the protection system is improved, the misjudgment caused by the detecting error of a single optical fiber is avoided, and the unnecessary personnel expense is reduced.