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In this study, a simple and selective biosensor has been introduced for determination of lactose by potentiometric method based on the catalytic behavior of kefir grains via modification with multi-walled carbon nanotubes (MWCNTs)/casein composite as indicator electrode and sat’d Ag/AgCl as reference electrode. To fabricate the indicator electrode, kefir grains were initially dried using a freeze dryer during 48 h time interval and powdered with an electronic grinder. Then ~ 0.15 g kefir powder was physically mixed with MWCNTs/casein with optimized weight ratio of 0.005/0.3 (w/w) and mixed with ~ 150 µL nujol oil. The generated paste was then packed inside a Teflon tube (i.d: 10 mm, height: 100 mm) and adopted as indicator electrode. Maximum potentiometric response was also observed at pH 7.0 and 0.51 M ionic strength, controlled using phosphate buffer (0.01 M) and NaCl (0.5 M), respectively. The linear dynamic range during lactose determination was ranged between 1.0×10-12 - 1.0×10-4 M with detection limit of 3.0×10-14 M based on extrapolation definition. Small hysteresis (lower than ~4%) was observed during sequential analyses of several lactose solutions with different molar concentrations. The response time based on the 90% of maximum response (t90) was estimated to be ~2 min. The validity of this method was evaluated via direct determination of lactose in some types of milk samples. This biosensor was applicable for indirect lactose determination via estimation of the end point of lactose-containing milk sample during titration by a dilute HCl solution (1.0 mM) as titrant.