The enhancement of bovine serum albumin (BSA) fluorescence detection using a hierarchical laser-induced periodic surface structure (LIPSS) at the TC4 titanium alloy substrate was experimentally demonstrated. The hierarchical structure, including microgrooves, submicrometer LIPSS, and nanoparticles, has been achieved by a femtosecond laser. Due to the surface plasmon polariton induced by the LIPSS and localized surface plasmon resonance induced by nanoparticles, the enhancement factor of BSA fluorescence detection reached 74. Further, a good linear relationship between the concentration of copper ion inside and BSA fluorescence intensity was found in the range between 5 and 35 μg/mL. Our method explores a simple, reproducible, and pollution-free technique for surface-enhanced fluorescence.