Objective: To investigate the failure in the hearing screening test among twin neonates in neonatal intensive care unit (NICU) and to further clarify the etiology of neonatal hearing impairment, thus to provide insights into prevention and early intervention. Methods: Automated auditory brainstem response(AABR), distortion product otoacoustic emission(DPOAE) and acoustic immittance were performed on 1452 neonates(including 130 twins) admitted in NICU from January 2015 to June 2018 and the risk factors including premature birth, hyperbilirubinemia, neonatal respiratory distress syndrome, etc. were analyzed retrospectively by univariate chi-square test and multivariate logistic regression analysis. Results: The incidence of C-section, premature birth, hyperbilirubinemia, low birth weight, very low birth weight, in-vitro fertilization, pregnancy-induced hypertension syndrome and formula or mixed feeding among twin neonates were significantly higher than those of singleton neonates ( P <0.05). The pass rates of the first-time AABR, DPOAE and acoustic immittance were significantly lower than singleton neonates. The proportion of twin neonates who failed the initial screening but recovered in the following test was as high as 72.86%. AABR pass rate was correlated with congenital heart disease, neonatal respiratory distress syndrome, C-section and (very) low birth weight. The pass rate of DPOAE was correlated with low birth weight and C-section. The pass rate of acoustic immittance was correlated with preterm birth, C-section, low birth weight, gestational diabetes and gestational hypertension. The pass rate of diagnostic ABR was associated with gestational diabetes. And the pass rate of diagnostic DPOAE was associated with maternal age ≥40 years old. Conclusion: The first-time hearing screening pass rate of twin neonates in NICU is lower than that of neonatal singleton. Most twin neonates who fail in the first screening test will recover. Preterm birth, neonatal respiratory distress syndrome, (very) low birth weight, congenital heart disease, gestational diabetes, pregnancy-induced hypertension syndrome, maternal age ≥ 40 years old and C-section are associated with the first-time failure in hearing screening tests among twin neonates, thus entailing close follow-up., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)