Introduction Deceased donor organ donation and transplantation in Sri Lanka is still in its early stages. Lack of organs is a significant problem at present. Further, availability of deceased donors lags behind those of live. One of the presumed reasons for this is poor knowledge and attitude among the health care staff and general public. The present study aimed to assess the knowledge and attitude of medical and nursing staff in surgical and medical intensive care units (ICU) in the National Hospital of Sri Lanka (NHSL) on deceased organ donation. Method A pre-tested, self-administered questionnaire on knowledge and attitude was used. Sample contained doctors and nursing officers of the ICUs of NHSL where potential donors are common. A percentage score was calculated with regard to knowledge and attitude. Ethical approval was obtained from the ethical committee of National Hospital of Sri Lanka. Results Sample contained 167 subjects; 110 (65.9%) nursing officers and 57 (34.1%) doctors. Majority were females (87.4%) and were in the 31-40-year age group (90, 53.9%). Of the sample, 110 (65.9%) had studied up to diploma, 44 (26.3%) up to basic degree and 13 (7.8%) were undergoing postgraduate training. Majority were Buddhists (102, 61.1%) followed by Muslims (20,12%) and Catholics (19,11.4%). About 40% (68) had ICU work experience of 1-5 years with the mean experience being 6.9 years (range 1-24) and 74.3% (124) were involved in the deceased organ donation process either in preparation or coordination. Of the sample, 87.4% (146) had a knowledge score of more than 50% with 4 (2.3%) achieving 100%. Nearly 75% (n, 127) acknowledged that they have adequate knowledge to address the public, however only 46.7% (78) were aware about the related legal procedures on deceased donor organ donation and transplantation in Sri Lanka. Majority (153,91.6%) possessed a positive attitude on organ donation while admitting the need for better awareness. There were no statistically significant associations between the age, designation, sex, level of education, religion or work experience with regard to knowledge and attitude (p>0.05). Conclusion There is a satisfactory knowledge and positive attitude among the medical and nursing staff in ICUs of NHSL on deceased organ donation and transplantation. However, knowledge on certain aspects need to be further improved specially on procedural and legal aspects, which may increase deceased organ donations in Sri Lanka.