Ambient noise affecting the activity and vocalization of animals has been an important environmental selection pressure. Many vertebrates use a number of strategies to avoid ambient noise, including avoiding noisy areas, prolonging call duration and increasing call frequency. However, some animals do not make any response to ambient noise. Here, we conducted a natural experiment to investigate the potential effects of ambient noise on the activity levels and echolocation calls in bats. We recorded the echolocation pulses and activity from bats while flying in twelve areas with different ambient noise levels whose equivalent continuous A-weighted sound pressure levels were 47.5 dB、47.7 dB、54.4 dB、54.9 dB、55.1 dB、55.1 dB、58.0 dB、60.6 dB、60.8 dB、62.5 dB、62.8 dB、67.3 dB respectively. A total of 1,017 pulses and 4,124 bat passes were analyzed at the twelve areas during all experimental trials over 12 successful nights. The results from linear regression models showed that changes in the number of bat passes and call parameters including pulse duration, starting frequency, peak frequency, ending frequency and bandwidth did not differ significantly among twelve areas (P > 0.05). The results may indicate that low frequency and low level (< 20 kHz, < 67.3 dB) ambient noise may have no significant effect on the echolocation calls and activity of bats producing high frequency calls.