Midazolam in aqueous solution, urine and serum were rapidly detected on site, based on surface-enhanced Raman spectroscopy (SERS) technology in this paper. The Raman spectra were recorded by a portable laser Raman spectrometer BWS415-785H with a wavelength of 785 nm. The spectrometer provided that the spectral measurement range would be 68～2 700 cm-1 and spectral resolution would be better than 3 cm-1. The output power was maintained 80 mW in the experiment with 5 s of integration time. First, the Raman spectra of Midazolam were calculated by density functional theory and compared with the experimental values. The possible characteristic peaks were identified. Then, using silver nanoparticles as active substrate, MgSO4 aqueous solution as neutral salt coagulants, the Raman peak at 689 and 827 cm-1 was selected as the characteristic peak on detection, the SERS detection of Midazolam was conducted. The limit of detection of Midazolam in aqueous solution samples is 6 μg·mL-1. In the range of concentrations of 5～40 μg·mL-1, the relationship of Raman peak intensity and concentration of Midazolam in aqueous solution can be expressed by a linear equation that was y=188.18x+743.05. The correlation coefficient was r=0.972, the recovery was 98.2%～107.2%, and the relative standard deviation (RSD) was 2.08%～3.25%. The limit of detection of Midazolam in urine samples is 20 μg·mL-1, In the range of concentrations of 15～125 μg·mL-1, the relationship of Raman peak intensity and concentration of Midazolam in urine can be expressed by a linear equation: y=59.78x-640.71. The correlation coefficient was r=0.958, the recovery was 96.9%～107.9%, and the RSD was 4.45%～5.75%. The limit of detection of Midazolam in serum samples is 20 μg·mL-1, In the range of concentrations of 15～125 μg·mL-1, the relationship of Raman peak intensity and concentration of Midazolam in serum can be expressed by a linear equation: y=30.81x+176.66. The correlation coefficient was r=0.963, the recovery was 94.2%～105.7%, and the RSD was 3.60%～4.41%. This method is rapid, accurate, non-destructive and easy to operate, and it lays a good foundation for Midazolam detection in the field.