To date, 25 million deaths have been caused by human immunodeficiency virus (HIV) infections. However, the lack of effective vaccines and drugs still leads to a high transmission rate of HIV. It was estimated that 2.7 million new infections occurred in 2011. Early and accurate diagnosis is essential to control the HIV transmission. Thus, many efforts have been made to explore a rapid and accurate method for HIV detection. The capabilities of antibody-based determination and nucleic-acid-based molecular diagnosis to detect HIV infections have been verified. Yet these methods have some intrinsic drawbacks. For example, antibody-based assays involve a lengthy process and may have an open-window issue while polymerase chain reaction (PCR)-based assays require delicate equipment and well-trained personnel. In this work, we present an integrated microfluidic system using a rolling circle amplification (RCA) assay which could automatically detect DNA fragment of HIV-1. With this approach, the HIV-1 could be detected automatically to reduce the risk of contamination with no human intervention. Additionally, the specially designed hook primer coated on the magnetic beads and a padlock probe both contribute to RNA extraction with higher specificity such that this developed system is capable of detecting HIV-1 from blood cells directly with improved accuracy. Successful demonstration for detection of HIV-1 DNA fragment has been achieved by using the proposed microfluidic platform.