Antifouling surfaces that could reduce nonspecific adsorption from a complex matrix are a great challenge in surface plasmon resonance (SPR) sensors. An antifouling surface made by the covalent attachment of DNA tetrahedron probes (DTPs) onto gold surfaces demonstrated superior antifouling property against protein and cell. DTP-modified Au (DTPs-Au) film for two single protein samples (1 mg/mL myoglobin, 48 mg/mL HSA) and five complex matrices (100% serum, 100% plasma, 9.85 × 108 red cells/mL, 5% whole blood, and cell lysate) had low or ultralow adsorption amounts (≤8.0 ng/cm2). More interestingly, DTPs-Au film could also avoid Au deposition on the surface in the process of the catalytic growth of gold nanoparticles (AuNPs). Thus, a low-fouling and sensitive SPR sensor for miRNA detection in a complex matrix was developed by integrating DTPs-Au film with the catalytic growth of AuNPs. Exploiting the amplification of catalytic growth of AuNPs, the detection limit was 0.8 fM toward target let-7a. Moreover, the SPR sensor revealed excellent selectivity and could distinguish let-7a from homologous family. More importantly, the SPR sensor could be feasible for determining miRNA in 100% human serum and cancer cell lysates, and the results of detecting miRNA from cancer cells were in excellent accord with the results obtained using qRT-PCR. This assay may have great potential as an miRNA quantification method in complex samples.