The aim of this study was to develop Thymoquinone (TQ) loaded PEGylated liposomes using supercritical anti-solvent (SAS) process for enhanced blood circulation, and greater radioprotection. The SAS process of PEGylated liposomes synthesis was optimized by Box-Behnken design. Spherical liposomes with a particle size of 195.6 ± 5.56 nm and entrapment efficiency (%EE) of 89.4 ± 3.69% were obtained. Optimized SAS process parameters; temperature, pressure and solution flow rate were 35 °C, 140 bar and 0.18 mL/min, respectively, while 7.5 mmol phospholipid, 0.75 mmol of cholesterol, and 1 mmol TQ were optimized formulation ingredients. Incorporation of MPEG-2000-DSPE (5% w/w) provided the PEGylated liposomes (FV-17B; particle size = 231.3 ± 6.74 nm, %EE = 91.9 ± 3.45%, maximum TQ release >70% in 24 h). Pharmacokinetics of FV-17B in mice demonstrated distinctly superior systemic circulation time for TQ in plasma. Effectiveness of radioprotection by FV-17B in mice model was demonstrated by non-significant body weight change, normal vital blood components (WBCs, RBCs, and Platelets), micronuclei and spleen index and increased survival probability in post irradiation animal group as compared to controls (plain TQ and marketed formulation). Altogether, the results anticipated that the SAS process could serve as a single step environmental friendly technique for the development of stable long circulating TQ loaded liposomes for effective radioprotection.