An electro-deposition approach was established to incorporate the gold nano-flakes onto the glassy carbon electrode in electrochemical cells (nano-Au/GC/ECCs). Using pulsed amperometric detection (PAD) without any gold oxidation for cleaning (non-oxidative PAD), the nano-Au/GC/ECCs were able to maintain their activity for oxidizing of carbohydrates in a normal alkaline medium. The reproducibility of peak area was about 2 relative standard deviation (RSD,%) for 6 consecutive injections. A dynamic range of carbohydrates was obtained over a concentration range of 5–80mgL−1 and the limits of detection (LOD) were of 2mgL−1 for fructose and lactose and 1mgL−1 for glucose and galactose. Moreover, the nano-Au/GC/ECC using the non-oxidative PAD was able to combine with the internal standard method for determination of lactose in fresh cow milk sample.