Carbon black (CB) is a nanostructured material widely used in several industrial processes. This nanomaterial features a set of remarkable properties including high surface area, high thermal and electrical conductivity, and very low cost. Several studies have explored the applicability of CB in electrochemical fields. Recent data showed that modified electrodes based on CB present fast charge transfer and high electroactive surface area, comparable to carbon nanotubes and graphene. These characteristics make CB a promising candidate for the design of electrochemical sensors and biosensors. In this review, we highlight recent advances in the use of CB as a template for biosensing. As will be seen, we discuss the main biosensing strategies adopted for enzymatic catalysis for several target analytes, such as glucose, hydrogen peroxide, and environmental contaminants. Recent applications of CB on DNA-based biosensors are also described. Finally, future challenges and trends of CB use in bioanalytical chemistry are discussed.