The aim of this study was to synthesize and characterize Cellulose Acetate (CA) porous scaffolds using the electrospinning technique associated with Hydroxyapatite (HA) and different concentrations of graphene oxide (GO), for advanced regenerative therapies application. The scaffolds were categorized into four distinct groups based on their composition: (1) Pure CA scaffolds; (2) CAHA scaffolds; (3) CAHAGO 1.0% scaffolds; (4) CAHAGO 1.5% scaffolds. Transmission Electron Microscopy (TEM) was used for the characterization of the nanocomposite. The scaffolds were analyzed by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM/EDS), and in vitro cell viability assays (WST). For the biological test analysis of Variance (two-way) was used, followed by Tukey’s post-test (α = 0.05). The TEM analysis allowed for the visualization of the deposition of HA on the graphene sheets, confirming the synthesis of the nanocomposite. XRD revealed the predominant presence of CaP phases in the CAHA, CAHAGO 1.0%, and CAHAGO 1.5% groups, underscoring the inherent mineral composition of the scaffolds. FTIR demonstrated cellulose characteristics and PO4 bands in the groups containing HA, confirming the effective incorporation of this material. Raman spectroscopy revealed distinct peaks in the GO groups, conclusively verifying the successful integration of graphene into the scaffold matrix. The micrographs showcased irregular pores filling the entire surface, arising from the intricate overlapping of fibers during scaffold formation. Importantly, all scaffolds exhibited excellent cell viability in the conducted assays. A proliferation process was observed in CAHA and CAHAGO 1.5% groups after 48 h ( p < 0.05). In conclusion, the scaffolds synthesized hold significant promise in the realm of tissue engineering and provide a fresh perspective on the possibilities for regenerative therapies.