One of the major concerns in target identification has been the need for new methods to detect target molecules in the native cellular environment. In conventional target identification, affinity-based pull down has been conducted using cell lysates. However, interactions in cell lysate do not reflect real endogenous interactions in living cells, and can produce false-positive or false-negative results. This study aimed to develop a new method of target protein identification in living cells. Targeting probes were conjugated onto magnetic nanoparticles (MNPs). After nanoparticle cellular uptake, identification and recruitment of target proteins were conducted in living cells, and the target protein was finally recovered under the magnetic field. As a proof-of-concept study, we developed a functionalized MNP (PTS1-MNP) to mimic a peroxisomal protein containing a synthetic peroxisomal targeting signal 1 (PTS1). The PTS1-MNPs were imported into human hepatoma HepG2 cells to recruit PTS1-receptor protein Pex5p. Successful peroxisomal translocation of PTS-MNPs was achieved via transient interaction with Pex5p. Pull-down of Pex5p in lysed or living HepG2 cells confirmed the selective recruiting functionality of synthetic PTS1. The specific detection of Pex5p before complete PTS1-MNPs translocation in living HepG2 cells further demonstrated the transient interaction between Pex5p and PTS1-MNPs. This is the first report showing the peroxisomal translocation of nanostructured materials in living cells. This approach can be applied as a new concept to study transient interactions and target identification or recruiting in living cells.