Herein, an economical, analytical and sensitive method was established for the fluorometric determination of urea using freshly prepared silver nanoparticles (Ag-NPs) in real urine samples. The standard addition and second-order derivative methods were selected for the ongoing research work to eliminate the possible effect of interferences in a real environment. In this work, Ag-NPs were prepared by reducing silver nitrate salt in the presence of 1,3-di-(1H-imidazole-1-yl) -2-propanol (DIPO) in an aqueous medium.