The invention relates to a genetically encoded fluorescent sensor for nicotinamide adenine dinucleotide, as well as methods of preparation and uses thereof. In one aspect, this invention relates to a sensor for detecting nicotinamide adenine dinucleotide, particularly, a recombinant fluorescent fusion protein sensor for detecting nicotinamide adenine dinucleotide. In one specific aspect, this invention relates to a recombinant fluorescent fusion protein sensor for detecting reduced nicotinamide adenine dinucleotide (NADH); in another specific aspect, this invention relates to a recombinant fluorescent fusion protein sensor for detecting oxidized nicotinamide adenine dinucleotide (NAD+); in yet another aspect, the invention relates to a recombinant fluorescent fusion protein sensor for detecting the ratio of reduced to oxidized nicotinamide adenine dinucleotide. This invention also relates to the method of preparing the sensors, and uses of the sensors in detecting NADH, NAD+, NADH/NAD+ ratio, screening drugs and measuring NADH metabolism.