Preterm birth (PTB) results in over 15 million early births annually and is the leading cause of neonatal deaths. There are no clinical methods currently available to evaluate risk of PTB at early stages in pregnancy; thus, a rapid diagnostic to analyze PTB risk would be beneficial. Microfluidic immunoaffinity extraction is a promising platform for preparing complex samples, such as maternal serum with PTB risk biomarkers. 3D printed microfluidic devices have advantages over conventional microfluidic systems including simple fabrication and potential for iterative optimization to improve designs. In this work, I developed immunoaffinity monoliths in 3D printed microfluidic devices modified with antibodies to enrich PTB biomarkers from human blood serum. I retained and eluted a peptide PTB biomarker in both buffer and blood serum using an immunoaffinity column. An additional three PTB biomarkers were also successfully extracted either from buffer or blood serum on single-antibody columns. Both polyclonal and monoclonal antibodies to PTB biomarkers were characterized by dot blots, biolayer interferometry, and surface plasmon resonance to determine their specificity and dissociation constants. I created multiplexed immunoaffinity columns to simultaneously enrich three PTB biomarkers from depleted human blood serum in a single extraction. This is the first demonstration of multiplexed immunoaffinity columns for PTB biomarkers in a 3D printed microfluidic device. My work is a key step towards the future development of 3D printed microfluidic devices for rapid PTB testing.