Origin–destination matrix is important for managing and controlling traffic as well as operating, designing, analyzing, and planning of transportation systems in the long period. It is challenging, expensive, and labour-intensive to estimate origin–destination matrices using conventional method. In comparison to the traditional method, estimating the origin–destination matrix using traffic count is significantly simpler and easier. The study aims to update the existing origin–destination matrix from traffic count by integrating CUBE software and genetic algorithms. It is usually beneficial to have coded algorithms that can be adjusted to the sort of data present in the research area. The base year origin–destination matrix for Kazhakoottam, located at Kerala, India was developed in CUBE software, through a four-step traffic assignment modelling process. The base year origin–destination matrix obtained from CUBE software was updated using genetic algorithm from traffic count. Genetic algorithm generates an ideal solution through repeatedly performing sequential procedures like reproduction, crossover, and mutation. A comparison with the observed link volume served to confirm the simulated link volume generated by the CUBE software. The average link volume error obtained for updated origin–destination matrix is 4.41%.