Currently, bio-based resources and modern techniques with cost-effective routes have lots of interests in the biological process. Herein, environmentally-friendly, cost-efficient, and biologically active folic acid (FA) modified layered double hydroxide (LDH) was fabricated. Then, to characterize the effect of the FA molecule on the LDH structure, we prepared pure carbonated LDH (LDH-CO32-) as well. Increasing in the interlayer space from 0.76 nm for LDH-CO32- to 1.28 nm for FA-modified-LDH (FA-LDH) approved the accomplishment of the intercalation process. Based on thermal analysis, the retained mass of FA-LDH was lower than the pure LDH; corresponding to the influence of organic segments. Subsequently, homogeneous nanocomposite (NC) films containing FA-LDH nano-sheets and chitosan (CS) were fabricated. Although the NC films showed fragile form, their char yield was improved (maximum 19%) compared to the neat CS film. Contact angle measurement indicated an improvement in the wettability behavior and more hydrophilic property for the prepared CS NCs. In addition, we examined the bioactivity potential of these NC films through the immersing of them in simulated body fluid with pH = 7.4, and the changes of pH for this solution were recorded for 30 days. The results of field emission scanning electron microscopy, Fourier transform infrared, and energy-dispersive X-ray analyses indicated the growth of hydroxyapatite [Ca10(OH)2(PO4)6] on the surface of the fabricated compounds. Accordingly, these NC films have great potential to be used in tissue engineering technology.