The objective of this work was to evaluate the influence of dichloromethane (CH2Cl2), potassium hydroxide (KOH) and phosphoric acid (H3PO4) in the treatment of biochar from guava seeds on conventional and non conventional techniques (ultrasound and microwave) for the immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption. The effects of the different treatments on the physical and chemical properties of the biochar were evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The immobilization of the BCL on the biochar was evaluated in hydrolysis reactions of olive oil. The conventional treatment of the biochar with KOH showed increased modification on the surface of the biochar, which presented a highly porous surface, and a greater activity for the immobilized biocatalyst compared with the other treatments. The results revealed the potential of biochar as a support novelty for the immobilization of enzymes and for their application in biocatalysis.