A new conjugated ladder polymer with a polyacene skeleton was synthesized in a Aldol-type condensation protocol between benzylic and aryl-ketone side groups of suitably functionalized single-stranded precursor polymers. The photophysical behavior of the new conjugated polyacene ladder polymer PAL comprising a polyacene skeleton has been investigated by steady-state and time-resolved photoluminescence spectroscopy in solvents of different viscosity and polarity, and by single-molecule spectroscopy (SMS). From the time-resolved photoluminescence experiments it is concluded that the excited state deactivation mainly results from energy transfer processes without significant conformational relaxation. When solutions in solvent mixtures of a good (THF) and a non-solvent (water) are studied, pronounced differences are seen between PAL and the well-known poly(para-phenylene) ladder polymer MeLPPP. Single molecule spectroscopy (SMS) reveals a remarkable heterogeneity in molecular shape, very much unlike MeLPPP, as determined by the polarization anisotropy and various photophysical properties. Several molecular models of PAL are characterized with density functional theory supporting this observation. We demonstrate that the PL lifetime, peak position, spectral width and vibronic intensity correlate with each other, implying that the polymer behaves as an intramolecular J-aggregate. Compared to MeLPPP, intramolecular energy transfer is not particularly efficient because of the high degree of disorder, which is also seen in the modest degree of photon antibunching and the pronounced temporal dynamics thereof due to the slow singlet–singlet annihilation.