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In this study, gold nanostars (AuNSs) were prepared by a facile and environmentally friendly method through the one-step reduction process with collagen as the stabilizing agent. The use of collagen, a highly biocompatible protein with many functional amines groups, can facilitate the simultaneous controlled synthesis and surface protecting of gold nanoparticles in one step. This synthetic process was operated in the aqueous solution of tetrachloroauric acid (HAuCl4) at room temperature, in which ascorbic acid serves as a reductive agent. The influence of collagen concentration (0.02-0.06 mM) on the morphology of AuNSs was carefully studied to clarify its dual roles as stabilizing and controlling agents for the growth of the particles. Besides that, by simply adjusting reaction components such as the molar ratio of ascorbic acid to HAuCl4 and pH value, the length of the AuNS tips was also controlled. This study could offer a novel modified approach in the controlled synthesis process of AuNSs with the biomolecules collagen. The resulting AuNSs were then characterized by ultraviolet-visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDS), zeta potential, Fourier transform-infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and circular dichroism (CD), as well as selected area electron diffraction (SAED). UV-Vis spectroscopy showed the formation of AuNSs with the maximum surface plasmon resonance peak at 600-639 nm. TEM results revealed that the average particle size of the AuNSs stabilized by the collagen ranged from 27.39 nm to 41.55 nm, depending on the experimental composition and the pH values. HRTEM, EDS, and SAED results prove a more precise insight into the formation of pure gold nanocrystals. Analysis of the current results may also help better understand the growth mechanism of AuNSs during the synthesis process in the presence of collagen. The Au concentration quantified by the inductively coupled plasma mass spectrometry (ICP-MS) technique after separating and decomposing with microwave-assisted digestion exhibits that the synthesis of AuNSs has a high yield of 88.62%. Additionally, the colloidal stability of AuNS-collagen against different NaCl concentrations, pH, temperatures, and storage time was also examined through UV-Vis spectroscopy. The investigation results reveal that AuNS-collagen remains stable in NaCl 2.0% (w/v), from mildly acidic to neutral pH (4-7), below the temperature of 40°C, and within 21 days postsynthesis. The AuNS synthesized by this eco-friendly method is promising for many potential applications in biomedical field.