This study develops a non-contact vibro-acoustic detection technique for measuring the defect quantity and determining the imperfection orientation surrounding a bone-implant interface. Acoustic excitation through a miniature loud speaker and vibration response measurement using a capacity-type displacement sensor are applied to accomplish this task to prevent the mass loading effect on the structure to be examined. The proposed non-contact excitation-response measurements are verified using a series of designated in vitro defect models， and the measured resonance frequencies (RFs) are used to discriminate interfacial structure variations. A finite element modal analysis is conducted to validate the measured RFs. Additionally， a prototype device is developed and applied to assess the osseointegration between dental implants and tibia in an in vivo animal model. A comparison of in vitro experimental results with numerical simulations shows that the RFs in the defective orientation are significantly smaller than those in the complete direction (p < 0.05)，and that the values decrease with increasing defect quantity (p < 0.05). Moreover，the defect depth affects RF variation. In the in vivo experiments，the RF levels in the lateral direction of the tibia are much higher than those in the axial direction (p < 0.05) of the tibia. The RF values in the axial direction for two implants have no significant difference (p = 0.552)，but the RFs in the lateral direction for implant 2 are higher than those for implant 1 (p < 0.05). The RF changes can be compared to assess osseointegration development. The proposed technique is promising for assisting dentists in the assessment of implant stability after surgery.