Real-time chemical and biological sensing in vitro is important for application in health and environmental monitoring. Thus, a more rapid and stable detection method is urgently needed. Herein, an immediate-stable real-time fluorescent immunosensor with a high response speed (∼100%, <1 s) and approximately zero steady-state error is constructed. The developed sensor is based on the MnO4--triggered in situ immediate-stable fluorogenic reaction between dopamine and orcinol monohydrate to produce azamonardine (DMTM). The obtained DMTM is identified and characterized by high-resolution mass spectrometry, 1H NMR spectroscopy, 13C NMR spectroscopy, and theoretical calculations. The present sensor achieves a highly sensitive detection of dopamine (DA) with a limit of detection (LOD) of 10 nM as well as alkaline phosphates (ALP) with an LOD of 0.1 mU/mL by using orcinol monohydrate phosphate sodium salt as a substrate. As a proof of concept, ALP-triggered fluorescence ELISA using cardiac troponin I (cTnI) as a model antigen target is further constructed. The developed real-time sensor achieves the detection of cTnI with an LOD of 0.05 ng/mL. Moreover, the sensor proposed by us is successfully applied to assess the cTnI level in clinical serum specimens and yields results consistent with those obtained by the commercial ELISA method. The immediate-stable real-time fluorescence immunosensor provides a promising and powerful platform for the trace detection of biomolecules in clinical diagnosis.