A new sandwich assay for tetracycline (TC) involving a DNA aptamer and antibody pair is demonstrated in conjunction with gold nanostar (GNS) enhanced surface plasmon resonance (SPR) to achieve detection in the low attomolar range. GNS particles were covalently functionalized with the antibody probe (antiTC) and integrated into a surface sandwich assay in conjunction with a SPR gold chip modified with the TC-specific aptamer. After it was demonstrated that both affinity probes can bind simultaneously to TC, optimization of the assay was performed using either antiTC only or GNS-antiTC conjugates to interact with aptamer/TC complexes present on the chip surface. Target concentrations as low as 10 aM could be detected using GNS-antiTC's, which was >103 times greater in performance than when using antiTC only. In addition, good selectivity was achieved with respect to other tetracycline derivative antibiotics, such as oxytetracycline (OTC) and chlortetracycline (CTC), both which are structurally similar to TC. As a demonstration of trace antibiotic analysis in environmental samples, the GNS enhanced sandwich assay was applied to analyze TC added to aliquots of local river water and the results validated by comparing to conventional high-performance liquid chromatography (HPLC) analysis.