Soluble all-trans polyacetylenes (PAs) are most ideal models to study the spectroscopic properties of conjugated polymers for precise control of their backbone configurations, resulting in semiconducting properties for optoelectronic applications. Herein, we report precise control of the backbone configuration and subsequent self-assembly of soluble PAs flanked with alkyl-fluoroalkyl side chains, using light and temperature stimuli respectively in solution. PAs with symmetric alkyl and unsymmetrical alkyl fluoroalkyl side chains were successfully polymerized by living cyclopolymerization with the olefin metathesis reaction mediated by third-generation Grubbs catalysts. Coil-to-rod-like conformational transition was observed for all of the PAs under ambient light due to photoisomerization, monitored by UV-vis spectroscopy and discussed in terms of the Huang-Rhys parameter. Enrichment of the trans configuration in the conjugated backbones opened up the possibility of aggregation between the isolated conjugated chains and further stabilization of rodlike conformers to realize the superior optoelectronic performance. Unlike symmetric alkyl-substituted PAs, at low temperature trans-rich PAs having unsymmetrical alkyl fluoroalkyl side chains exhibited strong aggregation due to interchain interactions with an extraordinary low optical band gap of 1.5 eV. It will be interesting to retain such ordered aggregates into thin solid films to be served as an ideal semiconducting macromolecular platform for future optoelectronic applications.