Using of enzymes in some industrial applications is still limited due to their instability under severe conditions, and relatively high production charge. Accordingly, this work deals with preparation and characterization of novel biocatalysts based on functionalized multi-walled carbon nanotubes (MWCNTs) with calix[8]arene in presence of N-ethyl-N-(3-dimethylaminopropyl) carbodiimide, (EDC) and glutardialdehyde (GA) crosslinkers. Among 5 honey isolates, the most potent levansucrase producer was identified based on 16S rRNA as Bacillus subtilis. Moreover, in case of using 4000 U/g enzyme concentration, the enzyme gave the highest immobilization yield (81%) onto uncrosslinked MWCNTs/calix[8]arene. The prepared materials were characterized using: FTIR, TEM and particle size distribution analysis. EDC had significant effect on the particle size (938 nm) relative to that in case of GA (746-784 nm) and un-functionalized MWCNTs (419 nm). Besides, the cytotoxicity of the immobilized enzyme could be minimized up to100 μg/mL concentration against BHK fibroblast normal cells in comparison with the free enzyme and MWCNTs. In general, all the prepared formulations improved the enzyme stability to great instance in comparison with the free enzyme.