A switchable fluorescence sensor based on a calix (Monapathi et al., 2021) [4]arene:Cu2+ complex (FLCX/Cu) has been developed for the detection of itraconazole (ITZ) with high sensitivity and specificity. For the development of the sensor, the selective complexation of a fluorescent calix (Monapathi et al., 2021) [4]arene derivative (FL-CX) with the Cu2+ ion causing fluorescence quenching was utilized. In addition, the sensor properties of the FLCX/Cu prepared were investigated. For this purpose, various substances (selected anions, cations, and drugs) with which ITZ can be found together were studied in an aqueous solution. Limit of detection (LOD) and limit of quantification (LOQ) values were determined in the range of 1.00–60.0 μg/L as 3.34 μg/L and 11.1 μg/L for ITZ, respectively. Moreover, the real sample analyses were performed in human serum and tablet form. Furthermore, the effect of some possible serum contents on sensor performance was also studied. All these studies confirmed the development of a simple, precise, accurate, reproducible, highly sensitive, and very stable fluorescence sensor.