The invention discloses a regulation operation anti-misoperation checking method based on anti-misoperation rule source side maintenance, comprising the following steps: a regulation main station acquires an equipment wiring diagram, model information and an equipment point table file from a transformer substation, and constructs a corresponding equipment model and main-sub station equipment comparison relationship; the regulation main station collects the on-off real-time remote communication state of a temporary grounding line/net door/trolley from a transformer substation microcomputer five-prevention system; the regulation main station acquires anti-misoperation logic rules in real time from the transformer substation microcomputer five-prevention system, and establishes an anti-misoperation rule expert knowledge base; and the regulation main station carries out anti-misoperation checking on remote control based on the anti-misoperation rules. According to the invention, the anti-misoperation rules of the transformer substation microcomputer five-prevention system are called to the main station in real time, and the anti-misoperation rule expert knowledge base is established in the main station, so that the anti-misoperation rules are convenient to extend and modify, the maintenance workload is small, the safety and reliability of operation ticket, instruction ticket, preview and remote-controlled anti-misoperation checking of the regulation main station are enhanced, and the operation stability of the regional power grid is enhanced effectively.