In recent years, with the progress of nanotechnology, polymer materials and advanced manufacturing technology, emerging flexible electronic devices represented by flexible sensors are playing an increasingly important role in the fields of wearable, healthcare, Internet of Things terminal and so on. As the carrier of flexible electronic devices, the flexible substrates are of great significance to the mechanical reliability and electrical sensing performance of sensors. However, the high hydrophobicity caused by the dominant non-polar bonds on the flexible substrate surface restricts the deposition of functional materials on the surface, which results in the unstable interfacial bond between the substrate and the electrode\sensitive layer. Therefore, the surface modification of flexible substrates by ultraviolet-ozone (UVO) treatment has received extensive attention. In this study, we explored the rapid and accurate evaluation of the UVO treatment effect of the flexible substrate by near infrared (NIR) spectroscopy, aiming to characterize the modification effect from the level of group and molecule, which is an effective supplement to the contact angle measurement method. In particular, four kinds of flexible substrates, polydimethylsiloxane (PDMS), polyethylene terephthalate (PEN), polyethylene terephthalate (PET) and polyimide (PI) were modified by 1/2/5/10 minutes with UVO treatment, and the modification effects were characterized by NIR spectroscopy. Finally, the characterization analysis results were verified by the contact angle measurement. The NIR spectrum analysis showed that the UV energy was not enough to break the methyl (—CH₃) functional group and (O—Si—O) chemical bond in the flexible PDMS substrate, so the hydrophilic groups such as hydroxyl group and carboxyl group could not be introduced. For flexible PEN and PET substrates, the treatment effect of UVO was better than that of flexible PDMS substrates, and the treatment effect of flexible PET substrates was better than that of flexible PEN substrates. The reason may be that the naphthalene ring double-ring structure in the PEN substrate has a strong ultraviolet light absorption ability, which blocks most ultraviolet energy below 380 nm. For flexible PI substrates, UVO treatment can effectively introduce active groups such as hydroxyl (C—OH) and carboxylic acid (O—C=O), and the strength and number of these functional groups increase with the increase of modification time, so that the surface energy of PI substrates increases in a short time, the contact Angle decreases, and the wettability improves. The contact angle measurement results showed that the UVO treatment had no obvious effect on the flexible PDMS substrate (the contact angle decreased by 8.4%). The modification effect of flexible PET substrate (39.6% contact angle decline) was better than that of flexible PEN substrate (9.4% contact angle decline). UVO treatment was the most effective for the flexible PI substrate, since the contact angle decreased by 62.7%.