A rapid,green and highly sensitive excitation-emission matrix(EEM)fluorescence method was proposed for analysis of irinotecan(CPT-I l)in biological fluids including human plasma and urine samples of uncalibrated interferences with the aid of second-order advantage.Due to the serious spectral overlapping from biological matrices,the parallel factor analysis(PARAFAC)and the alternating normalization-weighted error(ANWE)have been recommended to perform directly calibration and overcome the problem which makes the traditional fluorospectrophotometer in trouble.Satisfactory results can be achieved.Furthermore,performance of the proposed method was evaluated based on figures of merit and some statistical parameters.The accuracy of both algorithms was validated by the elliptical joint confidence region(EJCR)test.The precision and repeatability were also investigated by the relative standard deviations(RSDs)of intra-day and inter-day.