Surface-enhanced Raman scattering (SERS) is a unique spectroscopy that can offer high-sensitive detection for many molecules. Herein, the Au particles deposited on carbon nanofiber-encapsulated magnetic Ni nanoparticles (NPs) (Ni@CNFs@Au) have been successfully synthesized for SERS measurements. The Ni@CNFs@Au substrates have the advantages of a high SERS sensitivity and good magnetic response. The Ni@CNFs could be directly obtained from CO2 hydrogenation on a Ni catalyst, which has been characterized as having rich carboxylic acid groups, graphitic structures, and a high surface area. The Ni@CNFs surface could effectively increase the density of hotspots during Au NP aggregation and influence the morphology of the Au nanostructures. The spherical shape, oval shape, and coral-like Au nanostructures were prepared on Ni@CNFs with various Au concentrations. Brunauer Emmett Teller, zeta potential, high resolution transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy measurements were used to characterize the Ni@CNFs@Au samples. The Au NPs deposited on the Ni@CNFs presented a suitable oval shape, and an average size of similar to 30-40 nm. The size allowed surprisingly ultrasensitive SERS detection of rhodamine 6G (R6G) with a resolution of approximately a single molecule under an excitation wavelength of 532 nm. Using 785 nm excitation, a low R6G concentration of similar to 1 x 10(-14) M was detected. Moreover, the Ni@CNFs@Au substrates could be rapidly magnetically separated after adsorption. Phenylalanine and tyrosine amino acids, which are associated with the liver disease, were examined using SERS with the Ni@CNFs@Au substrate. Ultralow concentrations of similar to 1 X 10(-11) M for phenylalanine and similar to 1 X 10(-13) M for tyrosine were clearly measured. The Ni@CNFs@Au substrates exhibited applicability as excellent SERS detection platforms that combine high-sensitivity and rapid magnetic separation for various adsorption molecules.