A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/tBu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/tBu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.