The detailed investigation of methylene blue (MB) dye with ovalbumin (OVA) was examined by multispectroscopic and computational techniques. The experimental results of emission spectral studies displayed that the quenching behaviour of OVA with MB dye is due to static quenching mechanism. Isothermal titration calorimetry experimental results suggested that the binding of MB dye became favoured with the aid of a favourable entropy contribution and negative enthalpy. The absorption and circular dichroism spectral experiments showed that the binding of MB dye prompted conformational modifications to the secondary structure of OVA protein. The computational studies have been used to predict the binding region and the stability of MB in OVA protein.