Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.