A highly selective method of lysozyme detection based on salicylicylphenylacetylene (1) fluorophores and graphene oxide (GO) was successfully developed. The fluorescent emission of 1 was decreased fivefold in the addition of GO (0.6 Abs) due to π-π and hydrophobic interaction between 1 and GO graphitic plane. In the presence of lysozyme (Lys), the non-emissive 1-GO became highly fluorescent as a result of strong interaction of 1 with the cationic Lys preventing the complexation between 1 and GO. The linear dynamic response of fluorescence intensity was observed in the Lys concentration range of 0–80µg/mL with the limit of detection of 65ng/mL. This label free technique was developed for quantitative determination of Lys in egg white sample with an analytical accuracy of less than 10% error.