In this paper, we have synthesized a new kind of poly(2-acrylamido-2-methylpropanesulfonic acid) with crown ether, and studied comparatively the single molecule force spectroscopy of the polymers with and without crown ether, in terms of desorption and elongation. The smooth desorption process enabled us to calculate the loading rate of the stretching process. For the two polymers, desorption forces were loading rate independent and ionic strength insensitive. Interestingly, the desorption force of the two polymers were undistinguishable in all conditions. These findings demonstrate: (1) the polymer chains adopt a train-like (flat) conformation at the interface with a high adsorption/desorption rate, (2) the spacer, which separates the charged group from the hydrophobic backbone and combines the two properties together, should account for the retained desorption force at high salt concentration, (3) the 20% less in linear charge density does not affect the desorption force remarkably since hydrophobic interaction dominates the adhesion force. In deionized water, PAMPS-co-Crown is less rigid than PAMPS since the uncharged side groups separate the charged groups and thus the repulsion between adjacent charged groups is reduced. As the salt concentration increased, the rigidity of the two polymers both decreased, suggesting that the external salt would screen the charges of the polyelectrolytes. The linear charge density and the ionic strength affect only the rigidity of single polyelectrolyte chain but not the adhesion force is another result of the “spacer effect”. This fundamental finding, which reveals the non-electrostatic origin of the interfacial interaction of polyelectrolytes, sheds new light on the understanding of polyelectrolytes, especially for those containing spacers.