As a postdoctoral researcher, Holmes worked on the total synthesis of Vitamin B with Albert Eschenmoser.
In 1972 he was appointed as a demonstrator to the University of Cambridge where he stayed for 32 years, ultimately as Professor of Organic and Polymer Chemistry, and Director of the Melville Laboratory for Polymer Synthesis where he oversaw the founding and initial decade of the Melville Laboratory.
Holmes early work at Cambridge expanded his interest in new techniques for synthesising small molecules that are biologically-active and practically-useful, including natural products (such as alkaloids) and peptidomimetics. In 1989, during systematic characterisation of a newly synthesised conductive polymer, Chloe Jennings working in Holmes research group observed that the polymer emitted light when a current was passed through it. An intensive period of research in Holmes group, and other polymer chemistry groups, led to the discovery of differently-coloured light-emitting polymers that spanned the visible colour spectrum. A subsequent collaboration with physicist Richard Friend and co-workers at Cambridges Cavendish Laboratory revealed the potential of these conjugated polymers for applications such as organic LEDs and rollable displays.
Friend and Holmes co-founded the company Cambridge Display Technology for commercial exploitation of these materials – an early success story of Silicon Fen.
In 2004 Holmes returned to his native Australia on a Federation Fellowship, to lead a group at the newly established Bio21 Institute. He has pursued the application of photovoltaic polymers to solar energy, and was instrumental in forming the Victorian Organic Solar Cell Consortium. He has also continued to develop new syntheses of novel, biologically-useful materials. An example is his groups' synthesis of phosphoinositides, amphiphilic phospholipids situated in the cell membrane, which collaborators at the Ludwig Institute for Cancer Research have used to probe the dynamics of signal transduction (intercellular signalling being an important component of many aspects of cell biology, including that of tumors).
Holmes has served on the editorial or advisory boards of numerous learned scientific journals, including Organic Letters, Chemical Communications and Angewandte Chemie. In 2006, his 1998 paper on electroluminescent polymers was the most highly cited paper in Angewandte Chemie's 120-year history. By August 2012 he had authored over 490 scientific papers and 52 patent applications.
In 2014 he was appointed as President of the Australian Academy of Science.