Cyclic guanosine monophosphate (cGMP) is a second messenger involved in olfactory signal transduction. cGMP levels modulate the sensitivity of olfactory neurons and are implicated in olfactory dysfunctions. Previous reports demonstrate that individuals with olfactory dysfunction exhibit lower cGMP levels in the nasal mucus, necessitating precise analytical quantitative methods. Micellar-based spectrofluorimetric methods improve the detection of low concentrations of fluorescent compounds by using micelles to enhance fluorescence and reduce quenching. This study presents a micellar-based spectrofluorimetric method using Tween 80 to enhance the fluorescence intensity of cGMP. cGMP shows a weak emission signal at 350 nm when excited at 255 nm, but the addition of Tween 80 significantly enhances its fluorescence intensity by forming micelles that solubilize the hydrophobic cGMP and reduce quenching effects. These micelles, created by Tween 80, encapsulate cGMP in a hydrophobic core, stabilizing it through hydrogen bonding and van der Waals interactions, which further enhances fluorescence. The method was evaluated according to ICH M10 guidelines and demonstrated a linear relationship for cGMP concentrations from 1 to 50 ng/mL, with high accuracy and precision across intra-day and inter-day analyses. It also showed high selectivity, accurately detecting cGMP even in the presence of other substances like albumin and lactoferrin. The method was used to determine cGMP levels in the nasal secretions of nine healthy volunteers and eleven patients with olfactory dysfunction, revealing that cGMP was present in both groups. However, the average cGMP levels were significantly lower in patients with olfactory dysfunction (9.89 ± 1.20 ng/mL) compared to healthy individuals (38.65 ± 3.18 ng/mL).