Uranium and its compounds are radioactive and toxic, as well as highly polluting and damaging the environment. Novel uranium adsorbents with high biosorption capacity that are both eco-friendly and cost-effective are continuously being researched. The non-living biomass of the fresh water green microalga Chlorella sorokiniana was used to study the biosorption of uranium from aqueous solution. The biosorption of uranium from aqueous solutions onto the biomass of microalga C. sorokiniana was investigated in batch studies. The results showed that the optimal pH for uranium biosorption onto C. sorokiniana was 2.5. Uranium biosorption occurred quickly, with an equilibrium time of 90 min. The kinetics followed a pseudo-second-order rate equation, and the biosorption process fit the Langmuir isotherm model well, with a maximum monolayer adsorption capacity of 188.7 mg/g. The linear plot of the DKR model revealed that the mean free energy E = 14.8 kJ/mol, confirming chemisorption adsorption with ion exchange mode. The morphology of the algal biomass was investigated using a scanning electron microscope and energy dispersive X-ray spectroscopy. The FTIR spectroscopy analysis demonstrated that functional groups (carboxyl, amino, and hydroxyl) on the algal surface could contribute to the uranium biosorption process, which involves ion exchange and uranium absorption, and coordination mechanisms. Thermodynamic simulations indicated that the uranium biosorption process was exothermic (ΔH = -19.5562 kJ/mol) and spontaneous at lower temperatures. The current study revealed that C. sorokiniana non-living biomass could be an efficient, rapid, low-cost, and convenient method of removing uranium from aqueous solution.