Lactoferrin (LAF), an iron-binding glycoprotein has several health benefits ranging from anti-cancer, anti-inflammatory and antimicrobial activities against large number of microorganisms and is present abundantly in milk. Enzyme linked immunosorption assay based analytical protocol has been often referred for the selective detection of LAF in real samples. Herein, we report a simple electrochemical methodology for the direct recognition of LAF in raw milk using a methylene blue (MB) immobilized iron impurity (2.1 wt.%) containing multiwalled carbon nanotube/nafion modified glassy carbon electrode system. It is interesting to observe that upon potential cycling of GCE/Nf-MWCNT in MB dissolved milk system, both MB and LAF got co-immobilized on the electrode surface, designated as GCE/Nf-MWCNT@MB-LAF and showed a selective bio-electrocatalytic reduction signal for H2O2 at −0.4 V vs Ag/AgCl in milk or pH 7 PBS system. From control electrochemical experiments such as loading MB in pH 7 phosphate buffer solution and simulated milk sample and effect of carbon material (activated charcoal, graphite nanopowder and functionalized MWCNT) on preparation of the LAF modified electrode, it is revealed that iron impurity in the MWCNT, MB and LAF of the modified electrode involved in the electron-shuttling process to facilitate the H2O2 reduction reaction. By utilizing this novel process, selective bio-electrocatalytic sensing of H2O2 with current sensitivity and detection limit values 0.035 μA μM−1 and 3.2 μM respectively and specific recognition of LAF in raw and water diluted milksamples have been successfully demonstrated. © 2017 Elsevier Ltd