Tunnelling induces stress change and displacement in the ground. The excavation of a new tunnel in stratified soil can trigger different patterns of stress redistribution, which may adversely influence nearby tunnels. Research on multi-tunnel interaction has mainly been performed on the assumption of a uniform ground. The effects of different soil stratifications on tunnelling interaction remain poorly understood. In this paper, three-dimensional numerical parametric studies verified by previous centrifuge tests were carried out to analyse the twin tunnelling effects in two-layered soil. An advanced hypoplastic constitutive model that can capture stress-, path-, and strain-dependency of soil behaviour is adopted. Numerical cases investigated include perpendicular twin tunnelling in two sand layers with different relative densities and the location of the interface between the two sand layers. It is revealed that larger settlements and a wider surface settlement trough occur when tunnelling in two-layered soil strata than in a uniform ground. This is because of the wider and larger soil arch induced in two-layered soil strata. The structural response including tunnel deformation, induced bending moment, and induced hoop stress of the existing tunnel can be greater when tunnelling in layered soil strata than in a uniform ground owing to larger stress relief. Moreover, the combination of bending moment and hoop stress can exceed the M−N failure envelope of the structure in layered soil. A conventional simplified assumption of a uniform ground can underestimate of the influence of new tunnel excavation on existing tunnels, resulting in unsafe designs.