This was a prospective study aimed at determining the diagnostic accuracy and role of Fine Needle Aspiration Biopsy (FNAB) in the diagnosis of peripheral lymphadenopathy in Calabar. It further aimed at determining the HIV serological status of affected patients as well as the enhancement of the development of skills and experience in aspiration and diagnostic cytology of lymph node in the University of Calabar Teaching Hospital (UCTH). Patients with persistent peripheral lymphadenopathy who presented to UCTH Calabar or adjourning hospitals were examined to role out ulcers. Fine needle aspiration biopsy for cytology using 21G needle on 5ml syringe, full blood count, erythrocyte sedimentation rate and Human Immunodeficiency Virus (HIV) screening tests were done to make initial diagnosis. Subsequently, lymph node biopsy and histology was carried out to determine the final diagnosis. Findings were analyzed to determine diagnostic accuracy and the usefulness of FNAB in the diagnosis of lymphadenopathy. The result obtained showed an overall diagnostic sensitivity of 87%, specificity of 97%, false positive rate of 8%, false negative rate of 6%, positive predictive value of 92%, negative predictive value of 94%, and an overall diagnostic accuracy of 93%. Twenty percent of all patients studied were found to be HIV positive, and the highest prevalence HIV infection was among the 2 subgroup with reactive lymph node hyperplasia. Facilities and experience in FNAB improved in UCTH during the study. It was concluded that fine needle aspiration biopsy is of high diagnostic accuracy and appropriate for routine diagnostic assessment of persistent peripheral lymphadenopathy in Calabar. Routine HIV diagnostic test was recommended for this group of patients.