A novel nanoprobe was developed for the detection of hyaluronidase as cancer marker, using fluorescein 5-isothiocyanate (FITC) as fluorescence indicator, hyaluronic acid (HA), and polyethyleneimine (PEI) functionalized graphene oxide (GO) as quencher. PEI was attached to GO through amide bonding, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. Aminated hyaluronic acid (HA) was then linked to GO-PEI (GOP) via the same route. Nuclear magnetic resonance, ultraviolet–visible–near infrared spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscope, and thermogravimetric analysis analyses confirmed the successful synthesis of GOP-HA. Finally, FITC was attached to GOP-HA through reaction between the isothiocyanate group of FITC and the amine group of aminated HA. The fluorescence of FITC is quenched in the resulted GOP-HA-FITC nanoprobe. Addition of hyaluronidase can cleave HA chains into small fragments, releasing FITC to emit fluorescence. Importantly, the nanoprobe is highly sensitive and selective, and can enter cells by specifically binding to the CD44 receptor on the surface of Hela cells. Therefore, GOP-HA-FITC nanoprobe could be used in targeted tumor cell imaging for the early diagnosis of cancers.