Several studies have shown the development of electrochemical biosensors based on enzymes immobilized in metal–organic frameworks (MOFs). Although enzymes have unique properties, such as efficiency, selectivity, and environmental sustainability, when immobilized, these properties are improved, presenting significant potential for several biotechnological applications. Using MOFs as matrices for enzyme immobilization has been considered a promising strategy due to their many advantages compared to other supporting materials, such as larger surface areas, higher porosity rates, and better stability. Biosensors are analytical tools that use a bioactive element and a transducer for the detection/quantification of biochemical substances in the most varied applications and areas, in particular, food, agriculture, pharmaceutical, and medical. This review will present novel insights on the construction of biosensors with materials based on MOFs. Herein, we have been highlighted the use of MOF for biosensing for biomedical, food safety, and environmental monitoring areas. Additionally, different methods by which immobilizations are performed in MOFs and their main advantages and disadvantages are presented.