To eliminate the effects of complex background signals and to enhance the accuracy of the diffusion coefficient measurement, derivative NMR spectroscopy with negligible loss of the spectral quality is introduced based on the customized Savitzky-Golay method and used to construct diffusion-ordered NMR spectroscopy (DOSY). The criterion of the method was established by simulations. The application of this method on mouse urine and serum showed that the accuracy and precision of diffusion coefficient measurements in a complex background were improved to enhance the identification of molecules. Graphical abstract Diffusion-ordered NMR spectroscopy is a powerful tool for analyzing complex mixtures. To improve the accuracy of diffusion coefficient measurement, the magnitude of complex derivative spectra is introduced as a post-processing method to eliminate the effects of background signals, broad signals, or distorted baseline. And thus, accurate estimate of the diffusion coefficient is ensured to enhance the molecule identification.