High lipophilicity, low bioavailability, and hostile gastrointestinal (GI) environment is the major concern in oral administration of thymoquinone (TQ). Herein, we developed chitosan (CS) modified solid lipid nanoparticles (SLNs) to improve oral bioavailability. SLNs were systematically optimized by using a three-factor three-level QbD approach. The optimized TQ-CS-SLNs were successfully evaluated for various in vitro and ex vivo experiments and further evaluated for its bioavailability in Wistar albino rats. The developed TQ-CS-SLNs showed particle size, polydispersity index, and drug entrapment in the range between 135.61 and 211.36 nm, 0.17–0.29, and 65.14–91.78 %. The zeta potential of TQ-CS-SLNs was found to be +12.52 ± 1.21 mV. Moreover, TQ-CS-SLNs showed a controlled release profile during 24 h of study. In addition, optimized TQ-CS-SLNs showed excellent mucoadhesion with 67.26 ± 2.18 % mucoadhesive efficiency. The intestinal permeation and confocal microscopy revealed higher permeation of TQ-CS-SLNs compared to TQ suspension. Furthermore, bioavailability results revealed that the optimized TQ-CS-SLNs represents many fold improved oral bioavailability of TQ compared to TQ suspension. Therefore, from the findings, it was concluded that the CS-SLNs could be an effective nanoplatforms for improved oral bioavailability of TQ.