Background and Aim: Universal newborn hearing screening program (UNHS) using otoacoustic emission (OAE) has been expanded dramatically. The aim of this study was to compare the characteristics of OAE in different time periods.Methods: In this cohort study, 54 well babies were tested using screening transient-evoked otoacoustic emission in 12, 24, 36, 48 hours and 1 week of their age in Tamin Ejtemaii hospital of Zahedan, Iran. Parameters of "pass" and "fail" level, duration of the test, repetition rate for getting definite result and cooperation level of the parents were analyzed.Results: The failing rate decreased markedly with age. The major part of this decrement concerned to the age of 12 to 24 hours (left ear: p=0.012 and right ear: p=0.057). Only for the age of 12 hours, the test repetition reduced the failing rate significantly (left ear: p=0.001 and right ear: p<0.001). The test duration reduced significantly with age increment and decreased from the 75 second in age 12 hours to 21 second in age of 24 hours. The test time median for 36, 48 hours and one week of age was 13, 8.4 and 14.3 second, respectively. Among the 54 parents requested to come back for retest one week later, 5 (9.3%) cooperated.Conclusion: Age effect on referral rate were confirmed in this study; high referral rate and poor parent cooperation were the main problems in universal newborn hearing screening program in Zahedan city.