The invention discloses a device and a method for on-line detection of the content of ammonia nitrogen in seawater, and aims to detect the content of ammonia nitrogen of a seawater solution on the basis of a fluorescent spectrophotometer method. The device comprises a first three-way electromagnetic valve, a second three-way electromagnetic valve, a peristaltic pump, a mixing heating device, a fluorescent flowing cuvette, a light source, a light receiver and a signal processing system. With the combination of a flow injection technique and the fluorescent spectrophotometer method, the two three-way electromagnetic valves and the peristaltic pump are controlled to feed samples in sequence, on-line detection of the content of ammonia nitrogen in the seawater is achieved, and the device and the method are high in integration degree, simple to operate, single in mixed agent, short in reaction time and high in effectiveness. The mixing heating device is adopted to heat a seawater sample solution and an OPA mixed reagent which are pumped into a flowing channel, and the seawater sample solution and the OPA mixed reagent are sufficiently mixed in the heating process, so that mixing and heating are integrated, the reaction security is ensured while the size is reduced, and the accuracy of detection results can be improved.