The muscle-type nicotinic acetylcholine receptors (nAChRs) are expressed at the neuromuscular junction, where they mediate the fast-synaptic response upon acetylcholine (ACh) release from motor neurons. The nAChR is one of the most studied ion channels and its gating mechanism has been described intensively. However, the reciprocal relationship between channel activation and agonist binding is still elusive. Herein, we adapted a confocal patch-clamp fluorometry (PCF) method to study state-dependent agonist binding and unbinding behavior in nAChRs. Functional properties of nAChRs were characterized in response to three well-known ligands, ACh, nicotine, and carbachol employing the whole-cell patch-clamp method. After comparing the ligands, ACh was selected to synthesize its fluorescently tagged derivative, fACh. fACh is an excellent congener of the native neurotransmitter agonist at nAChRs. The binding of fACh to nAChRs is specific and reversible. Moreover, fACh produces adequate fluorescence signal upon HeNe Laser excitation (543 nm) so that the agonist and its mediated current could be monitored simultaneously. A rapid and a slow phase of fluorescence increase of fACh during binding corresponds to the activation and the desensitized state of the channel, respectively. Interestingly, increased fluorescence data points during the slow phase of binding confer that the desensitized state has a higher affinity for ACh than the resting state. Further, two phases of fluorescence decrease during unbinding of fACh might indicate that the recovery of the channel is already completed, while unbinding is still ongoing at the second binding site of the channel. Present work demonstrated the feasibility of exploiting fluorescent agonists to shed light on the activation mechanisms of the nAChR and other members of the Cys-loop superfamily.