A facile and efficient strategy to prepare multicolor and surface-functionalizable conjugated polymer nanoparticles (PPVseg-COOH CPNs) was demonstrated. The CPNs with tunable photoluminescence colors and carboxylate groups were further covalently modified with a series of specific molecules such as streptavidin, IgG and poly(ethylene glycol) to show their generality for subsequent bioconjugation and biological applications. The streptavidin coating can significantly improve the photostability of the PPVseg-SA CPNs, which indicates that specific biomolecules such as streptavidin functionalization of multicolor PPVseg-COOH CPNs can be applied to achieve high optical stability of CPNs in various buffer solutions, metal ions for many biological applications. Furthermore, the resulted PPVseg-SA CPNs also show efficient labeling ability in specific cellular imaging. The synthetic methods present the feasibility and versatility for further developing surface-functionalizable CPNs probes with full-color tunability for biological imaging and bioanalytical applications.