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The objective of this study was to determine the physicochemical and nanostructural properties of the alkali pre-treated gelatin from 3 different body parts (skin, muscle, and bone) of cornet fish (Fistularia petimba). The maximum yield of protein and viscosity was obtained in bone than other parts and Fourier transform infrared (FTIR) spectroscopy was carried out to characterize the extracted gelatin. Prior to extraction, the proximate composition was investigated clearly and the percentage of protein (15.50–29%), ash (4.32–6.50%), moisture (71.85–79.20%), and lipid (0.36–1.38%) content were estimated. Morphology of alkaline gelatin extracted from skin, muscle, and bone were studied using atomic force microscopy (AFM). The AFM images represented the scanning was done at 1.9 μm, 510, and 110 nm for skin, muscle, and bone, respectively showed the separate individual aggregates and the nano size particles of 0.17 μm, 100, and 219 nm, respectively. Amino acids like glycine, proline, and hydroxyproline were found with highest level in all the body parts of the extracted gelatin using HPLC.