A surface molecular imprinting technique was utilized in the fabrication of an enantio-selective adsorbent based on amino-functionalized alginate microspheres for chiral resolution of ascorbic acid. Alginate microspheres were first strengthened via epichlorohydrin (ECH) covalent cross-linking then functionalized with amino groups through graft copolymerization of polyacrylamide (PAm) followed by Hofmann degradation. Surface molecular imprinting was then performed under mild conditions by ionic interaction between the surface incorporated amine groups and the template l-ascorbic acid enantiomers followed by cross-linking with glyoxal. l-Ascorbic acid enantio-selective adsorbent (LA-Alg) was finally obtained by removing the template molecules out of the cross-linked network formed on the surface of the modified alginate particles. The synthetic steps were monitored using elemental analysis and FTIR spectra. Also, the surface morphologies of the native unmodified alginate along with both imprinted and non-imprinted adsorbent particles were examined by SEM. Moreover, the crystalline profile and thermal properties of both native and modified samples were investigated using XRD spectra and thermogravimetric analysis (TGA), respectively. The effect of pH on the extraction process was studied and indicated that the maximum adsorption was obtained at pH 4. Also, adsorption isotherms over LA-Alg adsorbent displayed the best fit with Langmuir model with maximum adsorption capacity 116 ± 1 and 67 ± 1 mg/g with respect to both l- and d-ascorbic acid, respectively. Moreover, the chiral resolution experiment using batch technique indicated 72% enantiomeric excess.