Gold-coated horizontally aligned carbon nanotube (Au-HA-CNT) substrates were fabricated for surface-enhanced Raman spectroscopy (SERS). The Au-HA-CNT substrates, which are granular in nature, are easy-to-prepare with large SERS-active area. Enhancement factors (EFs) of ∼10(7) were achieved using the Au-HA-CNTs as substrates for rhodamine 6G (R6G) molecules. Maximum enhancement was found when the polarization direction (E-field) of the incident laser beam was parallel to the aligned direction of the HA-CNTs. Simulations using the finite-difference time-domain (FDTD) method were carried out for the granular Au-HA-CNT samples. Enhancement mechanisms and determination of EFs were analyzed. Biological samples, including (13)C- and deuterium (D)-labeled fatty acids and Coccomyxa sp. c-169 microalgae cells, were also measured using this SERS substrate. The limits of detection (LODs) of D- and (13)C-labeled fatty acids on the SERS substrate were measured to be around 10 nM and 20 nM, respectively. Significantly enhanced Raman signals from the microalgae cells were acquired using the SERS substrate.