Hydrogen produced by microorganisms has been considered as a potential solution for sustainable hydrogen production for the future. In the current study, an advanced real-time optimisation methodology is developed to maximise the productivity of a 21-day fed-batch cyanobacterial hydrogen production process, which to the best of our knowledge has not been addressed before. This methodology consists of an economic model predictive control formulation used to predict the future experimental performance and identify the future optimal control actions, and a finite-data window least-squares procedure to re-estimate model parameter values of the on-going process and ensure the high accuracy of the dynamic model. To explore the efficiency of the current optimisation methodology, effects of its essential factors including control position, prediction horizon length, estimation window length, model synchronising frequency, terminal region and terminal cost on hydrogen production have been analysed. Finally, by implementing the proposed optimisation strategy into the current computational fed-batch experiment, a significant increase of 28.7% on hydrogen productivity is achieved compared to the previous study.