The invention provides an online detection method and device for cyclic fuel injection quantity of an engine under the variable working conditions. The specific values of instantaneous intake pressure and instantaneous exhaust pressure to cylinder pressure are calculated according to dynamic intake pressure, dynamic exhaust pressure and dynamic cylinder pressure, and in combination with the instantaneous flowing areas at an intake valve and an exhaust valve, the temperature of working media on the intake side and the temperature of working media on the exhaust side, the instantaneous flows of gas entering a cylinder and gas exhausted out of the cylinder are calculated; the instantaneous flows of gas entering the cylinder and gas exhausted out of the cylinder are subjected to integration, so that the quantity of accumulated gas in the cylinder is obtained when the intake valve is closed; and according to the quantity of accumulated gas in the cylinder when the intake valve is closed currently and the excess air coefficient, the cyclic fuel injection quantity is calculated. The problem that so far, the cyclic fuel injection quantity of the internal combustion engine is difficult to accurately detect online under the transient working conditions is solved, and the nuance of the air intake and fuel injection quantity of each cylinder can be detected accurately.