We developed a fluorescent aptasensor for label-free detection of ochratoxin A (OTA) based on TdT- and DNA polymerase-assisted multisite strand displacement amplification. This aptasensor exhibits good specificity and high sensitivity with a limit of detection (LOD) of 0.18 ng mL-1, and it can be further applied for the accurate quantification of OTA in complex real samples, holding promising applications in the field of food safety.