Research on biohydrogen production via fermentation process has shown a tremendous progress for the past few years. As biohydrogen production is being established, the purification of biohydrogen should consider the process flow for future application. This paper presents an experimental study of biohydrogen purification using two-stage chemical absorption. The research work focuses on carbon dioxide (CO2) removal, which is a major unwanted fermentation gas product via activated methyldiethanolamine (MDEA) and caustic (NaOH) in two-stage chemical absorption. The experiment was conducted at low pressure of 1 bar and normal room temperature of 29 °C using a ratio of 1:1 of CO2:H2 standard gas mixture as the feed. In the first stage, 40 wt. % MDEA was activated by using piperazine (PZ) with the concentration between 2 and 10 wt. %, whereas 20 wt. % NaOH was used in the second stage. It was found that 6 wt. % of PZ was required to fully activate 40 wt. % MDEA, which resulted in 79% CO2 removal. To improve CO2 removal, a gas distributor and wire mesh packed were used to create gas bubbles at higher geometrical surface. The experimental study successfully removed 99.59% of the total CO2, producing >99 mol% hydrogen gas purity from the second stage that used 20 wt. % NaOH.