Due to their unique structures and multifunctionalities, two-dimensional (2D) nanomaterials have aroused increasing interest in the construction of the novel biointerfaces for biosensing applications. Efforts in constructing novel biointerfaces led to exploit the more versatile and tunable graphene-like 2D nanomaterials (e.g. graphitic carbon nitride, boron nitride, transition metal dichalcogenides, and transition metal oxides) with various structural and compositional characteristics. This review highlights recent efforts in the design of graphene-like 2D nanomaterials and their derived biointerfaces and exploitation of their research on fluorescent sensors and a series of electrochemical sensors, including amperometric, electrochemiluminescence, photoelectrochemical and field-effect transistor sensors. Finally, we discuss some critical challenges and future perspectives in this field. •Graphene-like 2D nanomaterials are promising in designing functional biointerfaces.•We review graphene-like 2D nanomaterial-based electrochemical sensors.•We review graphene-like 2D nanomaterial-based fluorescent sensors.•Rational design of novel graphene-like nanomaterial-based biointerfaces is summarized.