The heavy bio-oil produced from biomass pyrolysis was used as carbon source to prepare porous biochar by one-step template method for methylene blue adsorption in wastewater. The effects of different templates and mass ratios on characteristic of porous biochar were studied. The methylene blue adsorption characteristics of porous biochar were investigated, and that of activated carbon was also discussed for comparison. The result showed calcium citrate was the most suitable template agent with the highest yield and the best microstructure of biochar. The specific surface area and micropore volume first increased and then decreased with the increasing mass ratio of template to bio-oil and carbonization temperature, reaching the maximum value of 922 m2/g and 0.182 cm3/g at 800 °C when the mass ratio was 2:1. The methylene blue adsorption capacity increased along with the increase of specific surface area and pH value. The adsorption capacity of porous biochar reached the maximum value of 411 mg/g at pH of 12, which was 28% higher than that of activated carbon. The adsorption process was consistent with Langmuir isothermal adsorption model and pseudo second-order kinetic model. This paper provides a new method to prepare effective adsorbent from heavy bio-oil for methylene blue removal.