In this study, gold nanoparticles (AuNPs) were synthesised using green chemistry to decorate multi-walled carbon nanotubes (MWCNTs) made from walnut shells transmission electron microscopy, field-emission scanning electron microscopy (FESEM), atomic force microscopy and fourier transforms infrared spectroscopy were used to diagnose MWCNTs and AuNPs. MWCNT-COOAu, MWCNT-COO and MWCNT-Au were diagnosed by Raman, energy dispersive X-ray analysis and FESEM. The effect of AuNPs, MWCNT-COO, MWCNT-COOAu and MWCNT-Au on pure and serum alkaline phosphatase (ALP) enzyme activity was studied in vitro using the enzyme-substrate 4-nitrophenyl disodium orthophosphate. For pure enzymes, Vmax slightly increased as the concentration of MWCNT-Au, MWCNT-COOAu and MWCNTCOO increased, whereas the Vmax values decreased as the concentration of AuNPs increased. The inhibition type for all NPs varied. For serum ALP enzyme, the Vmax values for Au-based NPs decreased as the concentration of NPs increased. The Vmax values exceeded the standard value at the concentrations of 25, 50 and 75 ppm for MWCNT-Au and MWCNT-COOAu, whereas the Vmax values increased over the standard value for all concentrations of AuNPs.