Abstract: A one-step nested PCR primers set and a kit modified with locked nucleic acid for detecting African swine fever virus are provided, relating to the field of molecular biology. It includes an outer primer pair, an inner primer pair and a probe. Upstream and downstream primer sequences of the outer primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. Upstream and downstream primer sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively. A sequence of the probe is shown in SEQ ID NO: 5. Based on the principle of nested PCR, the Tm value and stability of outer primers are improved by designing two pairs of nested primers and simultaneously modifying the outer primers with locked nucleic acid, and two independent circular nested amplification are performed, which has high sensitivity and specificity, easiness in operation and less cross contamination.