To enable the visualization of the distribution and dynamics of intracellular biomolecules and thereby understand the mechanisms of intracellular bioreactions, we developed a specific functional nanoprobe through the combination of a well-designed, cytocompatible phospholipid polymer and molecular beacons (MBs). A water-soluble, amphiphilic phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl tetra(ethylene glycol) methacrylate] (PMBS), was synthesized and conjugated with MBs to form nanoprobes via a chemical reaction between the ester group of N-hydroxysuccinimide and the amine group of the MBs. Surface tension measurements indicated that the polymeric nanoprobes had different conformations in aqueous solution, specifically at a concentration of 1.0 mg/mL. The PMBS, containing the large, hydrophobic BMA, formed polymer aggregates. The carcinoma cells used to test the probes remained 100% viable after incubation with PMBS-MB probes. The polymeric nanoprobes demonstrated not only a high target specificity but also resistance to nonspecific adsorption of proteins compared with unconjugated MBs and were able to penetrate the cytoplasm of the cells, allowing the live imaging of mRNA. In summary, MPC polymer-MB nanoprobes have great potential for practical application for the noninvasive monitoring of intracellular biomolecules and bioreactions in real time.