The challenge of economical separation of tiny microalgal cells from diluted solutions restricts their industry commercialization as promising biosorbents. In this study, The freshwater self-flocculating microalga Tetradesmus obliquus AS-6-1 was used as biosorbent to recover gold from wastewater. Maximum Au(III) adsorption capacity was obtained at optimal conditions of 0.1 g L−1 biomass, pH 2.0, 25 °C within 30 min for an initial concentration of 5 mg L−1. The higher maximum adsorption capacity (qm) and Langmuir constant (b) for T. obliquus AS-6-1 indicated its potential as efficient adsorbent for gold recovery. Detailed surface characterization demonstrated that polysaccharides excreted from the self-flocculating microalga were responsible for the better adsorption performance of T. obliquus AS-6-1. Flocculating activity results showed that T. obliquus AS-6-1 could efficiently settle down at the bottom by bio-flocculation within 20 min. The regenerated microalgae in the funnel reactor retained high adsorption efficiency of > 97% in the first two adsorption/desorption cycles. The results from this study firstly demonstrated that the self-flocculating microalga not only benefited its biomass recovery by its bio-flocculating property but also improved its potential for gold recovery from wastewater.