Based on traditional fluorescence spectroscopy and metal nanoparticles-enhanced fluorescence technology, this research explores a method of improving the accuracy and resolution of cholesterol detected by fluorescence spectroscopy in human whole blood solution. In experiment, an adult blood with silver nanoparticles is radiated by a laser pulse with wavelength of 407 nm, the fluorescence enhancement effect of cholesterol in blood is studied. The results show that, colloidal silver nanoparticles can enhance the fluorescence intensity of cholesterol in human blood with low concentration significantly. With the increase of the amount of silver colloids, the enhanced efficiency of fluorescence peaks at different positions increases first, and then decreases. However, the strongest enhanced efficiency of fluorescence peaks is different corresponding to different amount of silver colloids. According to the experimental results and the distribution of cholesterol molecules and silver nanoparticles in solution, molecular spatial distribution model is established by theoretical analyses, and the optimal distance for efficient fluorescence enhancement between cholesterol molecules and silver nanoparticles is calculated, the range is 12.19～25 nm, and the result is in good agreement with the theoretical values in other literatures. In summary, the fluorescence intensity of cholesterol in human blood can be enhanced by colloidal silver nanoparticles, and the results also provide a valuable reference on improving the sensitivity and accuracy of cholesterol detection.