We report a simple analytical procedure combining near-infrared (NIR) spectroscopy with multivariate analysis to detect the saccharification efficiency of pretreated rice straw. Three types of sample preparation methods were tested to develop a powerful calibration model, with the disk sample used as the standard protocol. From the spectra dataset of NaOH-treated biomass, we obtained a good calibration for the saccharification ratio and some major structural components by partial least-squares regression. Adding dataset from hot water and dilute sulfuric acid pretreatments to NaOH sample dataset, an acceptable calibration model to predict the saccharification ratio as well as the glucose, xylose, and lignin contents was generated. NIR has a great potential for rapid screening of saccharification efficiency of pretreated biomass, which would allows us to control the quality of processing toward better bioethanol production.