This paper concerns spectrofluorometric analysis of putrescine using orthophthaladehyde as a fluorophore in aqueous alkaline medium. Wavelengths of excitation and emission in acid, neutral, and alkaline media were different. There is a maximum intensity of fluorescence in alkaline medium 24 h after complexation compared with other media. Putrescine and orthophthaladehyde are used at an equimolar ratio, and the product is kinetically stable in alkaline medium. Calibration curves obtained gave limits of detection and quantification of 39 and 65 ng/mL, respectively. The correlation coefficient obtained in alkaline medium was 0.992 at pH 12. Results obtained largely showed a good reproducibility of our method.