In this paper, we investigate the existence of positive solutions for the eigenvalue problem of nonlinear fractional differential equation with generalized p-Laplacian operatorD0+β(ϕ(D0+αu(t)))=λf(u(t)),0<t<1,u(0)=u′(0)=u′(1)=0,ϕ(D0+αu(0))=(ϕ(D0+αu(1)))′=0,where 2<α⩽3,1<β⩽2 are real numbers, D0+α,D0+β are the standard Riemann–Liouville fractional derivatives, ϕ is a generalized p-Laplacian operator, λ>0 is a parameter, and f:(0,+∞)→(0,+∞) is continuous. By using the properties of Green function and Guo–Krasnosel’skii fixed-point theorem on cones, several new existence results of at least one or two positive solutions in terms of different eigenvalue interval are obtained. Moreover, the nonexistence of positive solution in term of the parameter λ is also considered.