Background: The World Health Organization (WHO) declared COVID-19 as a pandemic on the 11^th of March 2020. Since then, many efforts are being carried out to contain the virus. Knowledge and attitude of people should be directed towards strict preventive practices in order to halt the spread of the virus. The aim of the current cross-sectional study is to assess the knowledge, practice and attitude of healthcare workers (HCWs) of National Institute of Laboratory Medicine and Referral Centre, Dhaka, Bangladesh using a structured questionnaire. Methods: A cross-sectional study among 139 healthcare persons was performed between April 2020 and May 2020 at National Institute of Laboratory Medicine and Referral Centre. A systematic random sampling strategy was carried out and the data was collected through a self-administered questionnaire of the knowledge, attitude and practice of healthcare workers regarding COVID-19. Results: About 87.2% of eligible subjects completed and returned the questionnaire had a mean age of (30.1±6.1) years, most of them were male (75.53%), the highest percentage of HCWs were medical technologists (53.23%) and the majority of them had less than 5 years' experience (45.3%). A total of 96.4 % of the respondents were aware of coughing and sneezing etiquette, 93.52% followed hand washing steps but only 70% knew the duration of the hand hygiene procedure, while 89.92% uses face mask when go outside. The main sources of COVID-19 information were social media and the CDC/WHO website (38% and 23%, respectively). Conclusions: The majority of HCWs had good knowledge and positive attitude toward COVID-19. However, the level of some knowledge and attitude lower than that expected for their position level towards the virus. So, there is a need for awareness campaigns to improve their knowledge, in some aspects.