Polyether-ether-ketone (PEEK) is becoming a popular component of clinical spinal and orthopedic applications, but its practical use suffers from several limitations. In this study, irregular nano-porous monolayer with differently functional groups was formed on the surface of PEEK through sulfonation and nitrification. The surface characteristics were detected by field-emission scanning electron microscopy, atomic force microscopy, energy-dispersive X-ray spectrometry, water contact angle measurements and Fourier transform infrared spectroscopy. In vitro cellular behaviors were evaluated by cell adhesion, morphological changes, proliferation, alkalinity, phosphatase activity, real-time RT-PCR and western blot analyses. In vivo osseointegration was examined through micro-CT and histological assessments. Our results reveal that the irregular nano-porous of PEEK affect the biological properties. High-temperature hydrothermal NP treatment induced early osteogenic differentiation and early osteogenesis. Modification by sulfonation and nitrification can broaden the use of PEEK in orthopedic and dental applications. This study provides a theoretical basis for the wider clinical application of PEEK. a To obtain a uniform porous structure, PEEK samples were treated by concentrated sulfuric acid and fuming nitric acid (82-80%) with magnetic stirring sequentially. b Effects of nanopores on biological behavior of bMSCS.