A real-time label-free lateral offset spliced coreless fiber (CF) Mach-Zehnder interferometer (MZI) biosensor functionalized with hydrophobin Grifola frondosa I (HGFI) was proposed for the detection of cytokine tumour necrosis factor alpha (TNF-α). The nanolayer self-assembled on the optical fiber surfaces by HGFI rendered the immobilization of probe TNF-α antibody and recognition of antigen TNF-α. Trifluoroacetic acid was utilized to remove the HGFI layer from the glass surface, which was validated by field emission scanning electron microscopy (FESEM) and water contact angle (WCA). Results demonstrated that the processes of HGFI modification, antibody immobilization and specific antibody detection can be monitored in real time. The proposed biosensor exhibited good specificity, repeatability and low detection limit for TNF-α, extending its application in inflammation and disease monitoring.