Air-bone gap (ABG) is an important indicator of hearing status after myringoplasty. A number of factors have been associated with ABG, but some patients still have ABG without identifiable cause. This study aimed to evaluate the relationship between tympanic membrane (TM) vibration using laser Doppler vibrometry (LDV) and ABG after myringoplasty. Between January 2013 and January 2015, 24 patients with ABG of unknown cause after myringoplasty were enrolled at the Beijing Tongren Hospital. Thirty normal controls were recruited from the hospital staff. All patients underwent primary overlay myringoplasty. Pre- and postoperative air conduction (AC) and bone-conduction (BC) thresholds, and ABG were measured. Umbo velocity transfer function (UVTF) for vibration of TM was measured with LDV. Air conduction thresholds were significantly reduced after myringoplasty (all P < .05), while BC thresholds were not significantly changed (all P > .05). ABG was significantly reduced after myringoplasty (all P < .05). Air-bone gap was correlated with UVTF at 1.0 kHz (r = -0.46; P = .024). For patients with UVTF >0.08 mm/s/Pa, ABG was correlated with UVTF (r = -0.56; P = .029). For post-myringoplasty ABG without readily observable causes, there was a significant relationship between ABG and TM vibration. These results provide new insights in the understanding of this relationship and may help explain ABG after myringoplasty when there are no clear contributing factors.