The main objective of this work was to investigate the effect of glycerol at different compositions on physical properties, morphology and water absorption of protein based thermoplastic from fish waste. Fish waste containing fish head, bones and fins were collected from local fish product processing plant before further cooking, pressing, drying and milling. Compound formulation was done using twin screw extrusion and compression moulding. Samples at different glycerol compositions (Control, 20 pph and 35 pph) were characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and water absorption. The addition of glycerol showed two broad bands between 1500-1700 cm−1 and 3000-3500 cm−1 which correlates with the amount of α-helix and β-sheets structure of the protein. The fracture surfaces at control changed from uneven surfaces with large particles to smooth surfaces. Plastic deformation was observed at control and 20 pph but was absence at high glycerol content. Increasing the glycerol content was found to influence the water resistance properties of fish protein by reducing the absorption capability due to amount of α-helix and β-sheets structure.