During the production process of some copper clad laminate (CCL), the content of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane in prepreg can affect the cross-linking and curing degree of resin directly, and thereby affect the properties of CCL. In this article, near-infrared (NIR) spectroscopy combined with partial least square regression using high performance liquid chromatography as a reference method were used for the determination of 3,3,5,7,7-pentamethyl-1,2,4-trioxepane in the CCL production progress. 160 spectra of prepreg samples randomly formed the calibration set, and 50 spectra of prepreg samples formed the validation set. The value of the root mean square error of calibration (w/w) and root mean square error of prediction (w/w) were 0.011% and 0.013% for 3,3,5,7,7-pentamethyl-1,2,4-trioxepane content (%, w/w), and the calculation and validation of the regression equation resulted in high correlation coefficients of 0.99 and 0.98, respectively. The scatter plot value of calibration set which was obtained from the root mean square error of cross-validation (w/w) was 0.015%, and the regression equation resulted in high correlation coefficient of 0.98. The results of the paired t-test revealed that there was no significant difference between NIR and HPLC method. Thus, the results obtained in this study reflect that NIR could be used as a rapid, accurate, and simultaneous technique to determine 3,3,5,7,7-pentamethyl-1,2,4-trioxepane content of prepreg in the production process.