Aims: Calmodulin (CaM) is vital for the survival of Plasmodium knowlesi, a simian malaria parasite that infects both macaques and humans in Southeast Asia. To advance antimalarial drugs development targeting this protein, it is imperative to produce ample quantities of pure CaM for further research. Hence, this study aims to establish a robust strategy for the heterologous expression and purification of CaM from Plasmodium knowlesi (Pk-CaM). Methodology and results: First, we optimised the gene sequence of Pk-CaM for Escherichia coli expression, chemically synthesised it and integrated it into the pET28a plasmid. The optimised gene displayed a 45.15% GC content and a 0.81 codon-adaptation index, making it highly compatible with E. coli. Pk-CaM expression was assessed under various conditions, with the best results achieved at a post-induction temperature of 20 °C for 16 h, yielding a fully soluble protein. Subsequently, we purified the protein using Ni2+-NTA affinity chromatography and size-exclusion chromatography (SEC), obtaining 15 mg from 1 L of culture. The folding properties of purified Pk-CaM were analysed using far-UV circular dichroism (CD) spectroscopy, revealing a predominance of helical structures, both with and without Ca2+ ions. Binding to Ca2+ ions induced structural changes, increasing the helical content compared to when Ca2+ ions were absent. Conclusion, significance and impact of study The optimal conditions for the recombinant expression and purification of Pk-CaM in a correctly folded and functional form were successfully established in this study. This achievement provides a solid foundation for conducting further comprehensive research in the pursuit of novel antimalarial drugs.