Zwitterionic cross-linked biodegradable nanocapsules (NCs) were synthesized for cancer imaging. A polylactide (PLA)-based diblock copolymer with two blocks carrying acetylenyl and allyl groups respectively was synthesized by ring-opening polymerization (ROP). Azide-alkyne "click" reaction was conducted to conjugate sulfobetaine (SB) zwitterions and fluorescent dye Cy5.5 onto the acetylenyl-functionalized first block of the diblock copolymer. The resulting copolymer with a hydrophilic SB/Cy5.5-functionalized PLA block and a hydrophobic allyl-functionalized PLA block could stabilize miniemulsions because of its amphiphilic diblock structure. UV-induced thiol-ene "click" reaction between a dithiol cross-linker and the hydrophobic allyl-functionalized block of the copolymer at the peripheral region of nanoscopic oil nanodroplets in the miniemulsion generated cross-linked polymer NCs with zwitterionic outer shells. These NCs showed an average hydrodynamic diameter ( Dh) of 136 nm. They exhibited biodegradability, biocompatibility and high colloidal stability. In vitro study indicated that these NCs could be taken up by MIA PaCa-2 cancer cells. In vivo imaging study showed that, comparing to a small molecule dye, NCs had a longer circulation time, facilitating their accumulation at tumors for cancer imaging. Overall, this work demonstrates the applicability of zwitterionic biodegradable polymer-based materials in cancer diagnosis.