A fluorescent probe for the detection of penicillin in medicaments was fabricated with iodine-rhodamine B(RB) associate,as the fluorescence signal intensity of RB would be weakened by iodine(I-3) solution or even disappear,while penicillin alkaline hydrolysis product penicillium thiazole could reduce I-3to I-,making the system fluorescent signal reappear.The fluorescent probe was applied in the determination of penicillin in medicaments at an excitation wavelength of 365 nm and an emission wavelength of 580 nm.Results showed that there was a good linear relationship between fluorescence intensity change and penicillin concentration in the range of 0-6 μmol/L with a limit of detection(LOD) of 1.88×10-9 mol/L.The recoveries were in the range of 97.0%-106% with relative standard deviations(n=6) of 2.6%-3.1%.The proposed method was simple,rapid,accurate and reliable,and could be used for the analysis of penicillin in medicaments.