Urbanization has become one of the most important anthropogenic forces affecting the dynamics and functioning of ecosystems. Among these, artificial light at night (ALAN) has emerged as a significant environmental factor especially in urban areas. Previous studies has demonstrated that ALAN disrupts circadian rhythms, daily activity patterns, and sleep behavior, thereby affecting a wide range of phenotypic traits including morphology, physiology and behavior. Therefore, nighttime light has the potential to exert profound effects on insect behaviour and survival, which may in turn alter population dynamics and community structure. At the same time, species may differ in their vulnerability or resilience to urban stressors, potentially enabling certain species to colonize urban environments more successfully and thereby shaping urban ecosystems. However, studies examining the effects of ALAN on Drosophila species with different ecological backgrounds remain limited. Here we aimed to demonstrate the effects of ALAN exposure on activity pattern and sleep structure and compared among species with different ecological backgrounds. A total of nine Drosophila species were selected and categorized based on their primary habitat, consisting of three urban species, three rural species, and three generalist species. To simulate nighttime lighting conditions, we established two light regimes: a control treatment with light–dark(LD) cycle, and an ALAN treatment with light–dim(LDIM) cycle. All experiments were conducted under controlled temperature and humidity to isolate the effects of ALAN. Daily activity pattern and sleep behavior was quantified using the Drosophila Activity Monitoring (DAM) system. Individuals were placed in tubes, and activity was recorded every 1 minute for 24-hours. Sleep was defined as any period of at least 10 consecutive minutes without recorded activity. Under the control condition without nighttime light, all species exhibited a bimodal activity pattern with two peaks in the morning and evening, and showed a pattern in which the proportion of sleep increased during the night. By contrast, ALAN drastically altered activity and sleep patterns, largely eliminating the morning and evening activity peaks and reducing the proportion of sleep during the night. The effects of ALAN on activity and sleep differed among species. We discuss the relationship between each species’ ecological background and its susceptibility to ALAN.