In this paper, we have reported a rapid, simple, sensitive, straightforward, and validated method for the concentration determination of curcumin (CUR) in nanoliposomes and plasma using the spectrofluorimetry. For both nanoliposomal formulation and plasma, methanol was used as a solvent to extract the CUR. The excitation and emission wavelengths were set at 423 nm and 527 nm, respectively. The method validation was performed based on International Council for Harmonization (ICH) guidelines, Q2, in which parameters; such as, linearity, precision, accuracy and etc., were determined. The results showed that the calibration curve was linear for CUR concentrations of 0.05 to 0.5 μg /mL with a correlation coefficient of 0.9996. The limit of detection (LOD) and limit of quantification (LOQ) were 0.03 and 0.10 μg/mL, respectively. Liposomal CUR (15 mg/kg) was injected intravenously to mice, and at certain time intervals (1, 3, 6, and 24 h), blood samples were collected. The samples were extracted by methanol and CUR concentrations were detected using a fluorescence spectrophotometer. Results indicated the rate of liposomal formulation decline was slower than free CUR. The results of this study indicated that the validation method based on fluorimetry which was developed here is reliable for the detection of CUR in liposomal formulations and plasma.