We present 12CO J = 2-1 line observations of G54.1+0.3, a composite supernova remnant with a mid-infrared (MIR) loop surrounding the central pulsar wind nebula (PWN). We map an area of 12′×9′ around the PWN and its associated MIR loop. We confirm two velocity components that have been proposed to be possibly interacting with the PWN/MIR-loop; the +53 km s−1 cloud, which appears in contact with the eastern boundary of the PWN and the +23 km s−1 cloud, which has CO emission coincident with the MIR loop. However, we have not found a direct evidence for the interaction in either of these clouds. Instead, we detected an 5'-long arc-like cloud at +15-+23 km s−1 with a systematic velocity gradient of ~3 km s−1 arcmin−1 and broad-line emitting CO gas with widths (FWHM) of ≤7kms−1 in the western interior of the supernova remnant. We discuss their association with the supernova remnant.