This chapter presents an improved method for constrained optimisation of biochemical systems production. The aim of the proposed method is to maximise its production and, at the same time, to minimise the total amount of chemical concentrations involved in producing the best production. The proposed method models biochemical systems with ordinary differential equations. The optimisation process became complex for the large size of biochemical systems that contain many chemicals. In addition, several constraints as the steady-state constraint and the constraint of chemical concentrations also contributed to the computational complexity and difficulty in the optimisation process. This chapter considers the biochemical systems as a nonlinear equations system. To solve the nonlinear equations system, the Newton method was applied. Then, both genetic algorithm and cooperative co-evolutionary algorithm were applied to fine-tune the components in the biochemical systems to maximise the production and minimise the total amount of chemical concentrations involved. Two biochemical systems were used, namely the ethanol production in the Saccharomyces cerevisiae pathway and the tryptophan production in the Escherichia coli pathway. In evaluating the performance of the proposed method, several comparisons with other works were performed, and the proposed method demonstrated its effectiveness in maximising the production and minimising the total amount of chemical concentrations involved.