The objective of this research was to improve the protein extraction processes of Rastrelliger kanagurta (Indian mackerel) to generate protein isolate with enhanced bio-physico-chemical properties and gel-forming ability. To achieve this, two novel approaches were designed that utilized an additional alkaline separation step and were compared to a conventional process: acid solubilization → alkaline solubilization → pI and acid solubilization → pI → alkaline solubilization. The novel extraction designs resulted in a lower lipid content, lipid oxidation, and TCA-soluble peptides, as well as improving the color and sensory features of the refined proteins, which corresponded to the lowest total heme pigments (p < 0.05). Furthermore, the protein isolate recovered with the modified processes showed significant changes in biochemical properties (decreases in Ca2+-ATPase activity/reactive sulfhydryl content and an increase in surface hydrophobicity) and dynamic rheological behavior. As a result, by altering the extraction procedure it was possible to obtain improved gel characteristics such as gel strength, color, expelled moisture, and improved gel microstructure. Moreover, this study demonstrated that the gel network was partly stabilized by disulfide bonds, according to SDS-PAGE. Overall, this study demonstrates that by optimizing protein extraction procedures a considerable improvement in quality can be achieved and that an additional alkaline extraction after isoelectric point precipitation results in the optimized gel-forming ability of mackerel proteins.