The present study dealt with the use of hydrophobically modified polycations (N-acylated chitosan, Nac) stabilized iron oxide nanoparticles (IOPs) as a three dimensional (3D) nano-matrix for the controlled fabrication of hydroxyapatite (HAP). Among three different fatty acid chlorides (hexanoyl, octanoyl, and myristoyl chloride) modified chitosan (Nac-6, Nac-8, and Nac-14, respectively), we demonstrated the Nac-6-IOPs as a novel 3D nano-matrix for the controlled fabrication of HAP due to its well dispersibility and stability in aqueous medium (pH 7.4). Thermogravimetric analysis (TGA) estimated that an average two Nac-6 molecules could interact with one IOPs when the weight ratio of Nac-6 to IOPs was 0.16. Fourier transforms infrared (FT-IR) spectroscopic showed that the interaction of the polymer with IOPs was through the amide group of Nac-6. Transmission electron microscopy (TEM) revealed that the Nac-6-IOPs were well stable, uniform and mono-disperse in aqueous medium (pH 7.4), with average size of 10nm. X-ray diffraction (XRD) and FT-IR analysis showed the evidence of HAP formation on the surface of Nac-6-IOPs. Scanning electron microscope (SEM) equipped with energy dispersive X-ray (EDX) analysis showed that the HAP on the surface of 3D Nac-6-IOPs nanomatrix looks an interesting morphology like quasi-spherical structured with a significant rough surface and highly compact configuration. EDX data analysis confirmed that Ca/P ratio is in excellent agreement with the closer theoretical value of 1.67 for natural HAP. Our investigation for the growth of HAP on the surface of Nac-6-IOPs approved that the functional groups and the geometry of the nano-matrix play a key role for inducing and directing the growth of HAP crystals mimicking to that of natural apatite.