This work presents the synthesize methods and applications of water-soluble and uniform “graphene oxide/ phosphoramide ligands” (GO/L) nanocomposites by a cursory approach without any need for additional reductants. They are potential to provide much better biological properties, as enzyme immobilization, anti-bacterial and antifungal activity, in comparison to that of bare graphene oxide (GO) and phosphoraimides. In the provided study, an in-depth research of three GO based membranes, in which GO acts as the bed for the phosphoramide ligands, is presented. It is also proved that interlayer D-spacing of these membranes is wider compared to that of GO. These compounds were characterized by different analytical methods such as IR and NMR spectrums, AFM, XRD analyses and UV–visible. Additionally, the immobilization mechanisms of Acetylcholinesterase enzyme (AChE) by the GO and its new derivatives, the effects of new compounds as anti-bacteria and anti-fungi tools on Gluconacetobacter xylinus(G.xylinus) bacteria (an environmental bacteria) and Yeast cells were studied. the results showed that the GO/ L films had more potency as biological tools compared to bare GO and phosphoramide ligands. In comparison to previous works, this study aimed to introduce facile methods to synthesize powerful compounds which are active in several biological areas, more importantly, these compounds are not only more environmentally friendly ones than many presented compounds, but also are more economical and time saving. Such investigations would rich the understanding of the GO-based membranes, and also empowers future rational designs for a variety of applications such as food industry and drug design.