A protocol for construction of high-quality rice ribosome footprint library is provided. Rice ribosome/RNA complexes are isolated with a modified low ionic polysome extraction buffer. After nuclease digestion, rice ribosome footprints are extracted using SDS method followed by column purification. High-quality rice ribosome footprint library is constructed via key steps including rRNA depletion, end repair, 3′ adapter ligation, reverse transcription, circularization, PCR enrichment and several rounds of purification.