Objective To investigate the referral rate, prevalence and aetiology of neonatal hearing loss. Methods A total of 11,894 infants were screened by two-stage transient evoked otoacoustic emission testing. Those who failed were diagnosed by distortion product otoacoustic emission, 1000 Hz probe tone tympanometry and auditory brainstem response. The results of these tests were analysed by statistical software SPSS16.0. Results The initial referral rate was 17.36%. The rescreening referral rate was 21.29%. The referral rate of initial screening in maternity wards (15.37%) was lower than in neonatal intensive care unit wards (22%) (chi-square [chi(2)], P < 0.05). There were 68 cases (106 ears) diagnosed with hearing loss (incidence 0.571%). Of these, 31 cases were conductive, 16 cases were sensorineural, and 21 cases were mixed hearing loss. The prevalence of hearing loss was 12.92% (38/294) in the bilateral referred group and 5.00% (30/600) in the unilateral referred group. The moderate/severe hearing loss was 33.33% (10/30) and 86.84% (66/76), respectively (chi(2), P < 0.05). The causes of hearing loss included jaundice (24.56%, 14/57), infection (24.56%, 14/57), asphyxia (19.30%, 11/57), low birth weight (17.54%, 10/57) and other factors (14.04%, 8/57). Conclusion Bilateral referrals were more likely to suffer greater degrees of hearing loss than unilateral referrals. Jaundice, infection, asphyxia and low birth weight were the major aetiologies of neonatal hearing loss.