Since the beginning of the COVID-19 pandemic, much research has been conducted globally, but relatively few studies have been carried out in complex emergency settings that pose numerous operational challenges. We conducted a qualitative study to explore the barriers and enablers of a COVID-19 cohort study conducted in South Sudan and Eastern Democratic Republic of the Congo, to inform future research on COVID-19 and infectious diseases in humanitarian settings. We used a case study design embedded within the original prospective cohort study. Qualitative data was collected through four health facility assessments, 28 key informant interviews, and a focus group discussion. Data were analyzed using a manual thematic analysis approach and summarized against four primary themes: testing challenges and enablers, perceptions and attitudes towards COVID-19, national health system considerations, and study management considerations. Findings suggest most of the challenges affecting the cohort study were not specific to COVID-19 research but have been a feature of previous infectious disease research carried out in complex emergencies. However, the pandemic has exacerbated certain problems. The high proportion of travellers enrolled due to testing mandates, stigmatization of infected individuals linked to the heavy global focus on COVID-19, strained resources during waves of increasing infections, and remote management requirements all negatively impacted the cohort study. Factors that facilitated the research included proactive management, data quality oversight procedures, and strong collaboration with national health stakeholders. The global impact of COVID-19, its high public profile, and specific pandemic policies pose further operational challenges for research in already complex humanitarian settings. Future studies could plan mitigation measures that include flexibility in staffing and budgets, strategies to expand testing, and early partnerships with local organizations and health authorities.