In two-dimensional transitional metal dichalcogenides, tuning the spin-valley-layer coupling via changing layer numbers and stacking orders remains desirable for their application in valleytronics. Herein, six-point star-like MoSe2 nanoflakes simultaneously containing different atom registration regions from monolayer to bilayer with 2H and 3R stacking order were fabricated, and the valley polarizations were comparably investigated by circular polarized photoluminescent spectroscopy. The degree of valley polarization was detected to be about 12.5% in the monolayer and 10% in the 2H bilayer, but greatly upgraded to about 40% in the 3R bilayer MoSe2. This enhancement was attributed to the multiband spin splitting and generation of spin-dependent layer polarization for the 3R MoSe2 bilayer, which is well evidenced by our ab initio calculations of the energy band structures. Our results demonstrate that preparing TMD crystals with controllable stacking orders and interlayer coupling is a promising route to tune the valley index in TMDs for developing valleytronics technology.