In the present study, we prepared a gelatin nanofiber matrix using an electrospinning technique and cross-linked the nanofibers with 10 % glutaraldehyde vapors. The insoluble nanofibers were functionalized with bioactive molecules like biotin (1 %) and galactose (1 %) by adsorption and coelectrospinning. Surface morphology and fiber dimension were analyzed using atomic force microscopy. The amounts of biotin and galactose bound to the nanofibers before and after adsorption were quantified using high-performance liquid chromatography. Human larynx carcinoma (HEp-2) cell attachment, morphology and cytotoxic characteristics were studied using crystal violet staining and the MTT assay. Cell attachment and viability were highest in biotin- and galactose-embedded nanofibers compared to native nanofibers. Cytotoxicity was less with biotin- and galactose-embedded and adsorbed nanofibers compared to control nanofibers. Hence, we suggest that these biocompatible, nontoxic, biodegradable, functionalized nanofibers could be a potential candidate for application in tissue engineering and scaffold preparation.