One of the Most Interesting and Recent Insights into Biomimetic Scaffold Nano-Biomaterial is Smart Scaffolding with Targeted Drug Delivery Ability. in Recent Decades, the Use of Graphene-Based Materials, Such as Nano-Graphene Oxide (NGO), as a Drug Carrier with Amphiphilic Properties, Has Attracted Considerable Attention of Scientists and Researchers in This Field. in Addition, One of the Important Global Problems is Increased Vitamin D Deficiency, Particularly in Pregnant and Postmenopausal Women. Therefore, in This Work, by Considering Hydrophobic Properties of Vitamin D, We Attempted to Examine its Loading and Release Both in the Presence of Surfactant and Surfactant-Free NGO-Aqueous Solution. at First, NGO Powder Was Synthesized by the Modified Hummer's Method. after the Preparation of Vitamin D and Tween 80 (TW) Solution, They Were Added to NGO Aqueous Solution. Simultaneously, the Next Vitamin D and NGO Aqueous Solution Was Prepared in a Surfactant-Free Mode. in Order to Evaluate the Loading Content, Both Solutions Were Centrifuged, and their Supernatant Was Analyzed by UV-Visible Spectroscopy. Additionally, FTIR Spectroscopy Was Employed to Determine the TW 80 Effects on Vitamin D and NGO. the Results Have Shown that Vitamin D Loading in Surfactant-Free Solution Was Approximately 0% While in the Presence of TW 80 It Was 75.37% ± 4.12. Therefore, the Combination of Vitamin D, TW 80, and NGO Can Be a Suitable Candidate for Carrying Hydrophobic Drugs in Smart Scaffolding, Especially in Bone Tissue Engineering.