The invention discloses an enzyme-coupled nucleic acid-silver nanoprobe. The preparing method of the enzyme-coupled nucleic acid-silver nanoprobe includes the steps that 1, an oligonucleotide solution, a silver nitrate solution and a sodium borohydride solution are prepared respectively with a phosphate buffered solution serving as the solvent; 2, the oligonucleotide solution and the silver nitrate solution are taken and mixed evenly, the phosphate buffered solution is added and stirred, and the sodium borohydride solution is added and stirred; 3, a FeSO4 aqueous solution is added into the liquid obtained in the step 2, a D-amino acid oxidase aqueous solution is added, and the mixture is shaken evenly, wherein oligonucleotide is 5'-CCCTTAATCCCC-3'. The nanoprobe is applied to the fluorescence analysis method to detect D-amino acid in food, and the detection lower limit can reach 10 nM; the nanoprobe has the advantages of being high in analysis sensitivity and selectivity, easy and convenient to use and the like; compared with a traditional detection method, a sample does not need derivatization, operation is easy, cost is low, and D-amino acid can be rapidly and conveniently detected.